The project seeks to identify protective factors against disease through collaboration with people who have significant risk factors for disease that nevertheless do not manifest typical signs and symptoms. In a pilot study,  big data  was used to identify individuals with apparent resistance to severe genetic disease. [ 2 ] [ 3 ] [ 4 ]


This approach may seem weird, since the gene that is known to cause a genetic disorder could also be dealt with (head on) by just using overwriting the genetic code of this faulty gene with "good code" using gene therapy.


However, there is never just one version of "good code" (even people that do not have a disorder, the gene that is otherwise known to cause the defect can be present with different code). So rather than having to deal with these problems, Stephen Friend decided to use a  workaround  method (which consists of the approach noted above). [ 5 ]


Initially, the diseases the project looked at were 170 severe, Mendelian, disorders. [ 6 ]  However, the genetic data gathered from 600,000 people was not enough [ 7 ] [ a ] (only resilient individuals of 8 of the targeted diseases were found). The list of diseases it know look at is the following: [ 8 ]


DNA sequences from 589,306 people were used, obtained from  23andMe ,  Beijing Genomics Institute ,  Broad Institute  and others. [ 6 ]


Critics have argued that the researchers could not contact any of people to positively ensure that they were indeed healthy, despite having the disease mutation. Human geneticist Daniel MacArthur of the Broad Institute in Cambridge, Massachusetts still regards the study as “important as a proof-of-principle”. [ 9 ] [ 10 ]


In response to this criticism, Friend and Schadt have modified their Resilience Project by inviting new volunteers who agree to be recontacted to participate through a website [ 10 ]


In April 2020, the Resilience Project launched a participatory research study open to individuals in the USA.


Second Generation Multiplex Plus (SGM Plus) , is a  DNA profiling  system developed by  Applied Biosystems . It is an updated version of  Second Generation Multiplex . SGM Plus has been used by the  UK National DNA Database  since 1998.


An SGM Plus profile consists of a list of 10 number pairs, one number pair for each of 10  genetic markers , along with two letters (XX or XY) which show the result of the  Amelogenin  sex indicating test. Each number pair denotes the two  allele  values for the marker - one value is inherited from each of the subject's parents. If both alleles are the same, then only a single number, rather than a pair, is recorded.


The genetic markers (or  loci ) used by SGM Plus are all  short tandem repeats  (STRs). The markers used are: VWA, D8S1179, D21S11, D18S51, TH01, FGA, D3S1358, D16S539, D2S1338 and D19S433. Where a marker's designation begins with D, the digits immediately following the D indicate the  chromosome  that contains the marker. For example, D21S11 is on  chromosome 21 . SGM Plus also uses the  amelogenin  (amelo) sex-indicating test.


SGM Plus differs from SGM in that SGM does not use the markers D3S1358, D16S539, D2S1338 and D19S433.


SGM Plus has eight markers in common with  CODIS  FGA, TH01, VWA, D3S1358, D8S1179, D16S539, D18S51, and D21S11. It differs from CODIS in that it uses the additional markers D2S1338 and D19S433 and does not use the five markers CSF1PO, TPOX, D5S818, D7S820, D13S317. [ 1 ]


The primers are tagged with the following fluorescent dyes for detection under electrophoresis:


The primers for each locus are arranged on the dyes in the following order, from low molecular weight to large molecular weight:


The dyes to which each primer is attached differ from those of the original SGM DNA profiling system.


The SGM Plus profile of subject GT36865 from a  National Institute of Standards and Technology  paper is given below: [ 2 ]


An SGM Plus profile retrieved from a DNA database would just list the allele values: [ 4 ]


15,18; 6,9; 11,13; 22,22; 31,32.2; 14,17; 17,20; 11,12; 13,16.3; 15,16; XY


Each value is the number of  tandem repeats  within the allele. A non-standard repeat is designated by the number of complete repeat units and the number of base pairs of the partial repeat, separated by a decimal point.


The probability of identity (also known as the random match probability) is the probability that two individuals selected at random will have an identical genetic profile.


Applied Biosystems estimates the probability of identity for SGM Plus to be approximately 1 in 13 trillion for African-Americans and 1 in 3.3 trillion Caucasian Americans. [ 5 ]


The  Human Genetics Commission  has reported that the random match probability is in the region of 1 in a trillion. However it stated "When the SGM Plus profiling system was first introduced, there was agreement within the scientific community that identifications with match probabilities lower than one in a billion would not be quoted in the courts of law, so as to avoid overstating the value of the DNA evidence to take into account that match probabilities are only estimates, and to make sure that the figure used was one that was meaningful to non-specialists."
 [ 6 ]


The UK  Crown Prosecution Service  states "SGM Plus DNA profiling is very discriminating between individuals. The probability of obtaining a match between the profiles of two unrelated individuals by chance is very low, of the order of 1 in a billion. However, it has not yet been possible to carry out the required statistical testing to be able to quote this match probability, and in practice a more conservative chance match figure of 1 in 1,000 million is used." [ 7 ]


SingldOut  was an online  dating  platform that claimed to use  genetic testing  to identify potential relationship matches. [ 1 ]  The company marketed its intention to bridge the gap between digital networking and biological compatibility. [ 2 ] [ 3 ]  The service used the professional networking site  LinkedIn  as well as  DNA  testing company Instant Chemistry. [ 1 ]  Jana successfully wrote, orchestrated and implemented a highly targeted Public Relations campaign that resulted in national and international media acclaim valued at over $2M in earned media exposure. Jana Bayad launched the site in July 2014. [ 1 ] [ 4 ] [ 5 ] [ 6 ]  It was shut down in November 2015. [ 7 ] [ better source needed ]


Upon registering with SingldOut, members were sent a  DNA test  kit. [ 1 ] [ 8 ]  To assess the biological compatibility of its members, SingldOut claimed to examine  immune system  genes, saying that they play a role in attraction, [ medical citation needed ]  as well as  serotonin transporter  genes, claiming these play a role in determining how someone might react in certain situations. [ 1 ] [ medical citation needed ]  Results from the DNA test were then posted on the user's profile and compared with the results of other users. [ 9 ] [ 10 ]


The company credited genetic testing with the ability to “identify up to 40 percent of the chemistry of attraction between two people.” [ 11 ] [ medical citation needed ]


Autologous CD34+ enriched cell fraction that contains CD34+ cells transduced with retroviral vector that encodes for the human ADA cDNA sequence , sold under the brand name  Strimvelis , is a  medication  used to treat severe  combined immunodeficiency  due to  adenosine deaminase deficiency  (ADA-SCID). [ 1 ]


ADA-SCID is a rare inherited condition in which there is a change (mutation) in the gene needed to make an enzyme called adenosine deaminase (ADA). [ 1 ]  As a result, people lack the ADA enzyme. [ 1 ]  Because ADA is essential for maintaining healthy lymphocytes (white blood cells that fight off infections), the immune system of people with ADA-SCID does not work properly and without effective treatment they rarely survive more than two years. [ 1 ]


Strimvelis is the first  ex vivo  autologous gene therapy approved by the  European Medicines Agency  (EMA). [ 2 ]


Strimvelis is indicated for the treatment of people with severe combined immunodeficiency due to adenosine deaminase deficiency (ADA-SCID), for whom no suitable human leukocyte antigen (HLA)-matched related stem cell donor is available. [ 1 ]


The treatment is personalized for each person;  hematopoietic stem cell  (HSCs) are extracted from the person and purified so that only  CD34 -expressing cells remain. Those cells are  cultured  with  cytokines  and  growth factors  and then transduced with a  gammaretrovirus  containing the human  adenosine deaminase  gene and then reinfused into the person. These cells take root in the person's  bone marrow , replicating and creating cells that mature and create normally functioning adenosine deaminase protein, resolving the problem. [ 3 ] [ 4 ] [ 5 ]  As of April 2016, the transduced cells had a shelf life of about six hours. [ 6 ]


Prior to extraction, the person is treated with  granulocyte colony-stimulating factor  in order to increase the number of stem cells and improve the harvest; after that but prior to reinfusion, the person is treated with  busulfan  or  melphalan  to kill as many of the person's existing HSCs to increase the chances of the new cells' survival. [ 4 ] [ 5 ]


The most common side effect is pyrexia (fever). [ 1 ]


Serious side effects may include effects linked to autoimmunity (when the immune system attacks the body's own cells) such as  hemolytic anemia  (low red blood cell counts due to their too rapid breakdown),  aplastic anemia  (low blood cell counts due to damaged bone marrow),  hepatitis  (liver inflammation),  thrombocytopenia  (low blood platelet count) and  Guillain-Barré syndrome  (damage to nerves that can result in pain, numbness, muscle weakness and difficulty walking). [ 1 ]


Leukemia is a risk of treatment with Strimvelis. [ 7 ]


The treatment was developed at San Raffaele Telethon Institute for Gene Therapy and developed by  GlaxoSmithKline  (GSK) through a 2010 collaboration with Fondazione Telethon and Ospedale San Raffaele. GSK, working with the biotechnology company  MolMed  S.p.A., developed a manufacturing process that was previously only suitable for  clinical trials  into one demonstrated to be robust and suitable for commercial supply. [ 8 ] [ 9 ]


Strimvelis, the first ex vivo autologous gene therapy approved by the EMA, has demonstrated remarkable efficacy and safety in clinical trials for the treatment of ADA-SCID. [ 10 ]


In April 2016, a committee at the  European Medicines Agency  (EMA) recommended marketing approval for its use in children with  adenosine deaminase deficiency , for whom no matched HSC donor is available, on the basis of a clinical trial that produced a 100% survival rate; the median follow-up time was 7 years after the treatment was administered. [ 3 ]  75% of people who received the treatment needed no further  enzyme replacement therapy . [ 11 ]  Efforts had begun 14 years before. The total number of children treated was reported as 22 [ 12 ]  and 18. [ 13 ]  Around 80% of patients have no matched donor. [ 14 ]  Strimvelis was approved  [ 15 ]  by the  European Commission  on 27 May 2016.


As of 2016, [update]  the only site approved to manufacture the treatment was MolMed. [ 6 ]


In 2016, Strimvelis obtained Marketing Authorization in Europe while under GSK holding. [ 16 ]


In 2017, GSK announced it was looking to sell off Strimvelis, [ 17 ]  and in March 2018, GSK sold Strimvelis to Orchard Therapeutics Ltd.; as of that time there had been only five sales of the product. [ 18 ]


As of 2023, [update]  the product has been licensed in Iceland, Norway, Liechtenstein, and the UK. [ 16 ]


The condition affects about 14 people per year in Europe and 12 in the U.S. [ 19 ]


The price for the treatment was set at  €594,000 , twice the annual cost of  enzyme replacement therapy  injections. [ 20 ]   Enzyme replacement therapy  for ADA requires weekly injections and costs about  US$4.25 million  for one patient over ten years. [ 14 ]


Strimvelis  is the brand name. [ 3 ]  The common name is  autologous CD34+ enriched cell fraction that contains CD34+ cells transduced with retroviral vector that encodes for the human ADA cDNA sequence . [ 3 ]


Substrate reduction therapy  offers an approach to treatment of certain  metabolic disorders , especially  glycogen storage diseases  and  lysosomal storage disorders . In a storage disorder, a critical failure in a  metabolic pathway  prevents cellular breakdown and disposal of some large molecule. If residual breakdown through other pathways is insufficient to prevent harmful accumulation, the molecule accumulates in the cell and eventually interferes with normal biological processes. Examples of lysosomal storage disorders include  Gaucher's disease ,  Tay–Sachs disease ,  Sandhoff disease , and  Sanfilippo syndrome .


In a metabolic or genetic pathway, enzymes catalyze a series of reactions. Each enzyme is regulated or mediated by one gene through its RNA and protein products. At each phase in the pathway, enzyme activity catalyzes a reaction in which a precursor molecule (the  substrate ) is transformed into its next intermediate state. Failure of the metabolic pathway leads to accumulation of the substrate, with possible harmful effects. Substrate reduction therapy addresses this failure by reducing the level of the substrate to a point where residual degradative activity is sufficient to prevent substrate accumulation.


Supercow  (also  super cow  or  super-cow ) is a term used in the  dairy industry  to denote lines or individual animals that have superior  milk  production: that is, which produce more milk per day, or in some cases produce more fat per gallon of milk.  [ 1 ] [ 2 ]


Until recently, super-cows have been developed through  selective breeding  – either traditional breeding or, since the 1960s,  artificial insemination . Now the term tends to be applied to cows that have been genetically altered or whose genome has been studied in order to improve breeding. [ 3 ]  By 2023, [ when? ]  according to a news report "[i]n many countries, including the United States, farmers breed  clones  with conventional animals to add desirable traits, such as high milk production or disease resistance, into the gene pool". [ clarification needed ]  In that year, Chinese scientists reported the cloning of three so called "super-cows" with a milk productivity "nearly 1.7 times the amount of milk an average cow in the United States produced in 2021" and a plan for 1,000 such super-cows in the near-term. [ 4 ]


Therapeutic gene modulation  refers to the practice of altering the  expression of a gene  at one of various stages, with a view to alleviate some form of ailment. It differs from  gene therapy  in that gene modulation seeks to alter the expression of an endogenous gene (perhaps through the introduction of a gene encoding a novel modulatory protein) whereas gene therapy concerns the introduction of a gene whose product aids the recipient directly.


Modulation of gene expression can be mediated at the level of  transcription   by DNA-binding agents (which may be  artificial transcription factors ),  small molecules , or  synthetic oligonucleotides . It may also be mediated post-transcriptionally through  RNA interference .


An approach to therapeutic modulation utilizes agents that modulate endogenous transcription by specifically targeting those genes at the  gDNA  level. The advantage to this approach over modulation at the mRNA or protein level is that every cell contains only a single gDNA copy. Thus the target copy number is significantly lower allowing the drugs to theoretically be administered at much lower doses. [ citation needed ]


This approach also offers several advantages over traditional  gene therapy . Directly targeting  endogenous  transcription should yield correct relative expression of  splice  variants. In contrast, traditional gene therapy typically introduces a gene which can express only one transcript, rather than a set of stoichiometrically-expressed spliced transcript variants. Additionally, virally-introduced genes can be targeted for gene silencing by methylation which can counteract the effect of traditional gene therapy. [ 1 ]  This is not anticipated to be a problem for transcriptional modulation as it acts on endogenous DNA.


There are three major categories of agents that act as transcriptional gene modulators: triplex-forming oligonucleotides (TFOs), synthetic polyamides (SPAs), and  DNA binding proteins . [ 2 ]


Triplex-forming oligonucleotides (TFO) are one potential method to achieve therapeutic gene modulation. TFOs are approximately 10-40 base pairs long and can bind in the  major groove  in duplex DNA which creates a third strand or a triple helix. [ 2 ] [ 3 ]  The binding occurs at polypurine or polypyrimidine regions via Hoogsteen hydrogen bonds to the purine (A / G) bases on the double stranded DNA that is already in the form of the  Watson-Crick helix . [ 4 ]


TFOs can be either polypurine or polypyrimidine molecules and bind to one of the two strands in the double helix in either parallel or  antiparallel  orientation to target polypurine or polypyrimidine regions. Since the DNA-recognition codes are different for the parallel and the anti-parallel fashion of TFO binding, TFOs composed of  pyrimidines  (C / T) bind to the purine-rich strand of the target double helix via  Hoogsteen  hydrogen bonds in a parallel fashion. [ 3 ]  TFOs composed of  purines  (A / G), or mixed purine and pyrimidine bind to the same purine-rich strand via reverse Hoogsteen bonds in an anti-parallel fashion. TFO's can recognize purine-rich target strands for duplex DNA. [ 2 ]


In order for TFO motifs to bind in a parallel fashion and create  hydrogen bonds , the nitrogen atom at position 3 on the cytosine residue needs to be  protonated , but at  physiological pH  levels it is not, which could prevent parallel binding. [ 2 ]


Another limitation is that TFOs can only bind to purine-rich target strands and this would limit the choice of  endogenous  gene target sites to polypurine-polypyrimidine stretches in duplex DNA. If a method to also allow TFOs to bind to pyrimidine bases was generated, this would enable TFOs to target any part of the  genome . Also the human genome is rich in polypurine and polypyrimidine sequences which could affect the  specificity  of TFO to bind to a target DNA region. An approach to overcome this limitation is to develop TFOs with modified nucleotides that act as  locked nucleic acids  to increase the  affinity  of the TFO for specific target sequences. [ 5 ]


Other limitations include concerns regarding  binding affinity  and specificity, in vivo stability, and uptake into cells. Researchers are attempting to overcome these limitations by improving TFO characteristics through  chemical modifications , such as modifying the TFO backbone to reduce  electrostatic repulsions  between the TFO and the DNA duplex. Also due to their high molecular weight, uptake into cells is limited and some strategies to overcome this include  DNA condensing agents , coupling of the TFO to hydrophobic residues like  cholesterol , or cell permeabilization agents. [ 2 ]


Scientists are still refining the technology to turn TFOs into a  therapeutic product  and much of this revolves around their potential applications in antigene therapy. In particular they have been used as inducers of  site-specific mutations , reagents that selectively and specifically  cleave  target DNA, and as modulators of  gene expression . [ 6 ]  One such gene sequence modification method is through the targeting DNA with TFOs to active a  target gene . If a target sequence is located between two inactive copies of a gene, DNA ligands, such as TFOs, can bind to the target site and would be recognized as DNA lesions. To fix these lesions,  DNA repair  complexes are assembled on the targeted sequence, the DNA is repaired. Damage of the intramolecular recombination substrate can then be repaired and detected if resection goes far enough to produce compatible ends on both sides of the cleavage site and then 3' overhangs are ligated leading to the formation of a single active copy of the gene and the loss of all the sequences between the two copies of the gene. [ 4 ]


In model systems TFOs can inhibit gene expression at the DNA level as well as induce targeted mutagenesis in the model. [ 6 ]  TFO-induced inhibition of transcription elongation on endogenous targets have been tested on cell cultures with success. [ 7 ]  However, despite much  in vitro  success, there has been limited achievement in cellular applications potentially due to target accessibility.


TFOs have the potential to silence  silence gene  by targeting transcription initiation or elongation, arresting at the triplex binding sites, or introducing permanent changes in a target sequence via stimulating a cell's inherent repair pathways. These applications can be relevant in creating  cancer therapies  that inhibit gene expression at the DNA level. Since aberrant gene expression is a hallmark of cancer, modulating these endogenous genes' expression levels could potentially act as a therapy for multiple  cancer  types.


Synthetic polyamides are a set of small molecules that form specific hydrogen bonds to the  minor groove  of DNA. They can exert an effect either directly, by binding a regulatory region or transcribed region of a gene to modify transcription, or indirectly, by designed conjugation with another agent that makes alterations around the DNA target site.


Specific bases in the minor groove of DNA can be recognized and bound by small synthetic polyamides (SPAs). DNA-binding SPAs have been engineered to contain three polyamide amino acid components: hydroxypyrrole (Hp),  imidazole  (Im), and  pyrrole  (Py). [ 10 ]  Chains of these amino acids loop back on themselves in a hairpin structure. The amino acids on either side of the hairpin form a pair which can specifically recognize both sides of a  Watson-Crick base pair . This occurs through hydrogen bonding within the minor groove of DNA. The amide pairs Py/Im, Py/Hp, Hp/Py, and Im/Py recognize the Watson-Crick base pairs C-G, A-T, T-A, and G-C, respectively (Table 1). See figure for a graphical representation of 5'-GTAC-3' recognition by a SPA. SPAs have low toxicity, but have not yet been used in human gene modulation.


The major structural drawback to unmodified SPAs as gene modulators is that their recognition sequence cannot be extended beyond 5 Watson-Crick base pairings. The natural curvature of the DNA minor groove is too tight a turn for the hairpin structure to match. There are several groups with proposed workarounds to this problem. [ 8 ] [ 11 ] [ 12 ] [ 13 ] [ 14 ]  SPAs can be made to better follow the curvature of the minor groove by inserting  beta-alanine  which relaxes the structure. [ 10 ]  Another approach to extending the recognition length is to use several short hairpins in succession. [ 15 ] [ 16 ]  This approach has increased the recognition length to up to eleven Watson-Crick base pairs.


SPAs may inhibit transcription through binding within a transcribed region of a target gene. This inhibition occurs through blocking of elongation by an RNA polymerase.


SPAs may also modulate transcription by targeting a transcription regulator binding site. If the regulator is an activator of transcription, this will decrease transcriptional levels. As an example, SPA targeting to the binding site for the activating transcription factor TFIIIA has been demonstrated to inhibit transcription of the downstream 5S RNA. [ 17 ]  In contrast, if the regulator is a repressor, this will increase transcriptional levels. As an example, SPA targeting to the host factor LSF, which represses expression of the human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR), blocks binding of LSF and consequently de-represses expression of LTR [ 18 ]
.


SPAs have not been shown to directly modify DNA or have activity other than direct blocking of other factors or processes. However, modifying agents can be bound to the tail ends of the hairpin structure. The specific binding of the SPA to DNA allows for site-specific targeting of the conjugated modifying agent.


SPAs have been paired with the  DNA-alkylating moieties cyclopropylpyrroloindole [ 19 ]  and chlorambucil [ 20 ]  that were able to damage and crosslink SV40 DNA. This effect inhibited cell cycling and growth. Chlorambucil, a chemotherapeutic agent, was more effective when conjugated to an SPA than without.


In 2012, SPAs were conjugated to SAHA, a potent histone deacetylase (HDAC) inhibitor. [ 21 ]  SPAs with conjugated SAHA were targeted to Oct-3/4 and Nanog which induced epigenetic remodeling and consequently increased expression of multiple pluripotency related genes in mouse embryonic fibroblasts.


Designer zinc-finger proteins are engineered  proteins  used to target specific areas of  DNA . These proteins capitalize on the  DNA-binding  capacity of natural  zinc-finger  domains to modulate specific target areas of the  genome . [ 22 ]  In both designer and natural zinc-finger motifs, the protein consists of two  β-sheets  and one  α-helix . Two  histidine  residues on the α-helix and two  cysteine  residues on the β-sheets are bonded to a  zinc  atom, which serves to stabilize the protein domain as a whole. This stabilization particularly benefits the α-helix in its function as the DNA-recognition and -binding domain.  Transcription factor TFIIIA  is an example of a naturally-occurring protein with zinc-finger motifs. [ 23 ]


Zinc-finger motifs bind into the  major groove  of helical DNA, [ 23 ]  where the  amino acid  residue  sequence  on the α-helix gives the motif its target sequence specificity. The domain binds to a seven- nucleotide  sequence of DNA (positions 1 through 6 on the  primary strand  of DNA, plus positions 0 and 3 on the  complementary strand ), thereby ensuring that the protein motif is highly selective of its target. [ 22 ]  In engineering a designer zinc-finger protein, researchers can utilize techniques such as  site-directed mutagenesis  followed by  randomized trials  for binding capacity, [ 22 ] [ 24 ]  or the  in vitro recombination  of motifs with known target specificity to produce a library of sequence-specific final proteins. [ 25 ]


Designer zinc-finger proteins can modulate  genome expression  in a number of ways. Ultimately, two factors are primarily responsible for the result on expression: whether the targeted sequence is a  regulatory region  or a  coding region  of DNA, and whether and what types of  effector domains  are bound to the zinc-finger domain. If the target sequence for an engineered  designer protein  is a regulatory domain - e.g., a  promoter  or a  repressor  of  replication  - the binding site for naturally-occurring transcription factors will be obscured, leading to a corresponding decrease or increase, respectively, in  transcription  for the associated  gene . [ 26 ]  Similarly, if the target sequence is an  exon , the designer zinc-finger will obscure the sequence from  RNA polymerase  transcription complexes, resulting in a truncated or otherwise nonfunctional gene product. [ 22 ]


Effector domains bound to the zinc-finger can also have comparable effects. It is the function of these effector domains which are arguably the most important with respect to the use of designer zinc-finger proteins for therapeutic gene modulation. If a  methylase  domain is bound to the designer zinc-finger protein, when the zinc-finger protein binds to the target DNA sequence an increase in  methylation state of DNA  in that region will subsequently result. Transcription rates of genes so-affected will be reduced. [ 27 ]  Many of the effector domains function to modulate either the DNA directly - e.g. via methylation, cleaving, [ 28 ]  or recombination of the target DNA sequence [ 29 ]  - or by modulating its transcription rate - e.g. inhibiting transcription via repressor domains that block transcriptional machinery, [ 30 ]  promoting transcription with activation domains that recruit transcriptional machinery to the site, [ 31 ]  or  histone - or other  epigenetic -modification domains that affect  chromatin  state and the ability of transcriptional machinery to access the affected genes. [ 32 ]  Epigenetic modification is a major theme in determining varying expression levels for genes, as explained by the idea that how tightly-wound the DNA strand is - from histones at the local level up to chromatin at the  chromosomal  level - can influence the accessibility of sequences of DNA to transcription machinery, thereby influencing the rate at which it can be transcribed. [ 23 ]  If, instead of impacting the DNA strand directly, as described above, a designer zinc-finger protein instead affects epigenetic modification state for a target DNA region, modulation of gene expression could similarly be accomplished.


In the first case to successfully demonstrate the use of designer zinc-finger proteins to modulate gene expression  in vivo , Choo  et al. [ 26 ]  designed a protein consisting of three zinc-finger domains that targeted a specific sequence on a   BCR-ABL   fusion   oncogene . This specific oncogene is implicated in  acute lymphoblastic leukemia . The oncogene typically enables  leukemia  cells to proliferate in the absence of specific growth factors, a hallmark of  cancer . By including a  nuclear localization signal  with the tri-domain zinc-finger protein in order to facilitate binding of the protein to genomic DNA in the nucleus, Choo  et al.  were able to demonstrate that their engineered protein could block transcription of the oncogene in vivo. Leukemia cells became dependent on regular growth factors, bringing the cell cycle back under the control of  normal regulation . [ 26 ]


The major approach to post-transcriptional gene modulation is via  RNA interference  (RNAi). The primary problem with using RNAi in gene modulation is drug delivery to target cells. [ 33 ] [ 34 ]  RNAi gene modulation has been successfully applied to mice toward the treatment of a mouse model for inflammatory bowel disease. [ 35 ]  This treatment utilized liposome-based beta-7 integrin-targeted, stabilized nanoparticles entrapping short interfering RNAs (siRNAs). There are several other forms of RNAi delivery, including: polyplex delivery, ligand-siRNA conjugates, naked delivery, inorganic particle deliver using gold nanoparticles, and site specific local delivery. [ 36 ]


Designer zinc-finger proteins, on the other hand, have undergone some trials in the  clinical arena . The efficacy and safety of EW-A-401, an engineered zinc-finger transcription factor, as a  pharmacologic agent  for treating  claudication , a  cardiovascular  ailment, has been investigated in clinical trials. [ 37 ]  The protein consists of an engineered  plasmid DNA  that prompts the patient to produce an engineered transcription factor, the target of which is the  vascular endothelial growth factor-A (VEGF-A)  gene, which positively influences  blood vessel  development. Although not yet approved by the U.S.  Food and Drug Administration  (FDA), two  Phase I clinical studies  have been completed which identify this zinc-finger protein as a promising and safe potential therapeutic agent for treatment of  peripheral arterial disease  in humans. [ 38 ]


Transfection  is the process of deliberately introducing naked or purified  nucleic acids  into  eukaryotic   cells . [ 1 ] [ 2 ]  It may also refer to other methods and cell types, although other terms are often preferred: " transformation " is typically used to describe non-viral  DNA  transfer in  bacteria  and non-animal  eukaryotic  cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state ( carcinogenesis ) in these cells.  Transduction  is often used to describe virus-mediated gene transfer into prokaryotic cells. [ 2 ] [ 3 ]


The word  transfection  is a  portmanteau  of  trans-  and  infection .   Genetic  material (such as  supercoiled plasmid DNA  or  siRNA  constructs), may be transfected. Transfection of  animal cells  typically involves opening transient pores or "holes" in the  cell membrane  to allow the uptake of material. Transfection can be carried out using  calcium phosphate  (i.e.  tricalcium phosphate ), by  electroporation , by cell squeezing, or by mixing a  cationic   lipid  with the material to produce  liposomes  that fuse with the cell membrane and deposit their cargo inside.


Transfection can result in unexpected morphologies and abnormalities in target cells.


The meaning of the term has evolved. [ 4 ]   The original meaning of transfection was "infection by transformation", i.e., introduction of genetic material, DNA or RNA, from a  prokaryote -infecting virus or  bacteriophage  into cells, resulting in an infection. For work with bacterial and archaeal cells transfection retains its original meaning as a special case of transformation. Because the term transformation had another sense in animal cell biology (a genetic change allowing long-term propagation in culture, or acquisition of properties typical of cancer cells), the term transfection acquired, for animal cells, its present meaning of a change in cell properties caused by introduction of DNA. [ citation needed ]


There are various methods of introducing foreign  DNA  into a eukaryotic  cell : some rely on physical treatment (electroporation, cell squeezing,  nanoparticles , magnetofection); others rely on chemical materials or biological particles (viruses) that are used as carriers. There are many different methods of gene delivery developed for various types of cells and tissues, from bacterial to mammalian. Generally, the methods can be divided into three categories: physical, chemical, and biological. [ 5 ]


Physical methods include  electroporation ,  microinjection ,  gene gun ,  impalefection ,  hydrostatic pressure , continuous infusion, and sonication. Chemicals include methods such as  lipofection , which is a lipid-mediated DNA-transfection process utilizing liposome vectors. It can also include the use of polymeric gene carriers (polyplexes). [ 6 ]  Biological transfection is typically mediated by  viruses , utilizing the ability of a virus to inject its DNA inside a host cell. A gene that is intended for delivery is packaged into a replication-deficient viral particle. Viruses used to date include  retrovirus ,  lentivirus ,  adenovirus ,  adeno-associated virus ,  and  herpes simplex virus . [ citation needed ]


Physical methods are the conceptually simplest, using some physical means to force the transfected material into the target cell's nucleus. The most widely used physical method is  electroporation , where short electrical pulses disrupt the cell membrane, allowing the transfected nucleic acids to enter the cell. [ 5 ]  Other physical methods use different means to poke holes in the cell membrane:  Sonoporation  uses high-intensity ultrasound (attributed mainly to the  cavitation  of gas bubbles interacting with nearby cell membranes),  optical transfection  uses a highly focused laser to form a ~1 μm diameter hole. [ 7 ]


Several methods use tools that force the nucleic acid into the cell, namely:  microinjection  of nucleic acid with a fine needle; [ 5 ]   biolistic particle delivery , in which nucleic acid is attached to heavy metal particles (usually gold) and propelled into the cells at high speed; [ 8 ]  and  magnetofection , where nucleic acids are attached to magnetic  iron oxide  particles and driven into the target cells by magnets. [ 8 ]


Hydrodynamic delivery  is a method used in mice and rats, in which nucleic acids can be delivered to the liver by injecting a relatively large volume in the blood in less than 10 seconds; nearly all of the DNA is expressed in the liver by this procedure. [ 9 ]


Chemical-based transfection  can be divided into several kinds:  cyclodextrin , [ 10 ]  polymers, [ 11 ]  liposomes, or nanoparticles [ 12 ]  (with or without chemical or viral functionalization. See below).


DNA can also be introduced into cells using  viruses  as a carrier. In such cases, the technique is called  transduction , and the cells are said to be transduced.  Adenoviral  vectors can be useful for viral transfection methods because they can transfer genes into a wide variety of human cells and have high transfer rates. [ 2 ]  Lentiviral vectors are also helpful due to their ability to transduce cells not currently undergoing mitosis.


Protoplast fusion is a technique in which transformed bacterial cells are treated with lysozyme in order to remove the cell wall. Following this, fusogenic agents (e.g., Sendai virus, PEG, electroporation) are used in order to fuse the protoplast carrying the gene of interest with the target recipient cell. A major disadvantage of this method is that bacterial components are non-specifically introduced into the target cell as well.


Stable and transient transfection differ in their long term effects on a cell; a stably transfected cell will continuously express transfected DNA and pass it on to  daughter cells , while a transiently transfected cell will express transfected DNA for a short amount of time and not pass it on to daughter cells.


For some applications of transfection, it is sufficient if the transfected genetic material is only transiently expressed. Since the DNA introduced in the transfection process is usually not integrated into the nuclear genome, the foreign DNA will be diluted through  mitosis  or degraded. [ 5 ]  Cell lines expressing the  Epstein–Barr virus  (EBV) nuclear antigen 1 (EBNA1) or the SV40 large-T antigen allow episomal amplification of plasmids containing the viral EBV (293E) or SV40 (293T) origins of replication, greatly reducing the rate of dilution. [ 25 ]


If it is desired that the transfected gene actually remain in the genome of the cell and its daughter cells, a stable transfection must occur.  To accomplish this, a  marker gene  is co-transfected, which gives the cell some selectable advantage, such as resistance towards a certain  toxin . Some (very few) of the transfected cells will, by chance, have integrated the foreign genetic material into their genome. If the toxin is then added to the cell culture, only those few cells with the marker gene integrated into their genomes will be able to  proliferate , while other cells will die. After applying this selective stress (selection pressure) for some time, only the cells with a stable transfection remain and can be cultivated further. [ 26 ]


Common agents for selecting stable transfection are:


RNA can also be transfected into cells to transiently express its coded protein, or to study  RNA decay  kinetics. RNA transfection is often used in primary cells that do not divide.


siRNAs  can also be transfected to achieve RNA silencing (i.e. loss of RNA and protein from the targeted gene). This has become a major application in research to achieve " knock-down " of proteins of interests (e.g. Endothelin-1 [ 27 ] ) with potential applications in gene therapy. Limitation of the silencing approach are the toxicity of the transfection for cells and potential "off-target" effects on the expression of other genes/proteins.


RNA can be purified from cells after  lysis  or synthesized from free  nucleotides  either chemically, or enzymatically using an  RNA polymerase  to  transcribe  a  DNA  template.  As with DNA, RNA can be delivered to cells by a variety of means including  microinjection ,  electroporation , and  lipid-mediated transfection .  If the RNA encodes a  protein , transfected cells may  translate  the RNA into the encoded protein. [ 28 ]   If the RNA is a regulatory RNA (such as a  miRNA ), the RNA may cause other changes in the cell (such as  RNAi-mediated knockdown ).


Encapsulating the RNA molecule in  lipid nanoparticles  was a breakthrough for producing viable  RNA vaccines , solving a number of key technical barriers in delivering the RNA molecule into the human cell. [ 29 ] [ 30 ]


RNA molecules shorter than about 25nt (nucleotides) largely evade detection by the  innate immune system , which is triggered by longer RNA molecules.  Most cells of the body express proteins of the innate immune system, and upon exposure to exogenous long RNA molecules, these proteins initiate signaling cascades that result in  inflammation .  This inflammation hypersensitizes the exposed cell and nearby cells to subsequent exposure.  As a result, while a cell can be repeatedly transfected with short RNA with few non-specific effects, repeatedly transfecting cells with even a small amount of long RNA can cause cell death unless measures are taken to suppress or evade the innate immune system (see "Long-RNA transfection" below).


Short-RNA transfection is routinely used in biological research to knock down the expression of a protein of interest (using  siRNA ) or to express or block the activity of a  miRNA  (using short RNA that acts independently of the cell's  RNAi  machinery, and therefore is not referred to as siRNA).  While DNA-based vectors ( viruses ,  plasmids ) that encode a short RNA molecule can also be used, short-RNA transfection does not risk modification of the cell's DNA, a characteristic that has led to the development of short RNA as a new class of  macromolecular   drugs . [ 31 ]


Long-RNA transfection is the process of deliberately introducing RNA molecules longer than about 25nt into living cells.  A distinction is made between short- and long-RNA transfection because exogenous long RNA molecules elicit an  innate immune response  in cells that can cause a variety of nonspecific effects including  translation  block,  cell-cycle  arrest, and  apoptosis .


The innate immune system has evolved to protect against  infection  by detecting  pathogen-associated molecular patterns  (PAMPs), and triggering a complex set of responses collectively known as " inflammation ".  Many cells express specific  pattern recognition receptors  (PRRs) for exogenous RNA including  toll-like receptor  3,7,8 ( TLR3 ,  TLR7 ,  TLR8 ), [ 32 ] [ 33 ] [ 34 ] [ 35 ]  the RNA  helicase   RIG1 (RARRES3) , [ 36 ]   protein kinase R  (PKR, a.k.a. EIF2AK2), [ 37 ] [ 38 ]  members of the oligoadenylate synthetase family of proteins ( OAS1 ,  OAS2 ,  OAS3 ), and others.  All of these proteins can specifically bind to exogenous RNA molecules and trigger an immune response.
The specific chemical, structural or other characteristics of long RNA molecules that are required for recognition by PRRs remain largely unknown despite intense study.  At any given time, a typical  mammalian  cell may contain several hundred thousand mRNA and other,  regulatory long RNA  molecules.  How cells distinguish exogenous long RNA from the large amount of endogenous long RNA is an important open question in  cell biology .  Several reports suggest that  phosphorylation  of the 5'-end of a long RNA molecule can influence its  immunogenicity , and specifically that 5'-triphosphate RNA, which can be produced during viral infection, is more immunogenic than 5'-diphosphate RNA, 5'-monophosphate RNA or RNA containing no 5' phosphate. [ 39 ] [ 40 ] [ 41 ] [ 42 ] [ 43 ] [ 44 ]  However, in vitro-transcribed (ivT) long RNA containing a  7-methylguanosine cap  (present in  eukaryotic  mRNA) is also highly immunogenic despite having no 5' phosphate, [ 45 ]  suggesting that characteristics other than 5'-phosphorylation can influence the immunogenicity of an RNA molecule.


Eukaryotic mRNA contains chemically modified nucleotides such as  N 6 -methyladenosine ,  5-methylcytidine , and  2'-O-methylated  nucleotides.  Although only a very small number of these modified nucleotides are present in a typical mRNA molecule, they may help prevent mRNA from activating the innate immune system by disrupting  secondary structure  that would resemble double-stranded RNA (dsRNA), [ 46 ] [ 34 ]  a type of RNA thought to be present in cells only during viral infection.
The immunogenicity of long RNA has been used to study both innate and  adaptive immunity .


Inhibiting only three proteins,  interferon-β ,  STAT2 , and  EIF2AK2  is sufficient to rescue human  fibroblasts  from the cell death caused by frequent transfection with long, protein-encoding RNA. [ 45 ]  Inhibiting interferon signaling disrupts the positive-feedback loop that normally hypersensitizes cells exposed to exogenous long RNA.  Researchers have recently used this technique to express  reprogramming proteins  in primary human  fibroblasts . [ 47 ]


Gene therapy  utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses (sometimes called biological  nanoparticles  or viral vectors) and those that use naked DNA or DNA complexes (non-viral methods).


All  viruses  bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hacking the body's normal production machinery to serve the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected. Some types of viruses insert their genome into the host's cytoplasm, but do not actually enter the cell. Others penetrate the cell membrane disguised as protein molecules and enter the cell.


There are two main types of virus infection:  lytic  and  lysogenic . Shortly after inserting its DNA, viruses of the lytic cycle quickly produce more viruses, burst from the cell and infect more cells. Lysogenic viruses integrate their DNA into the DNA of the host cell and may live in the body for many years before responding to a trigger. The virus reproduces as the cell does and does not inflict bodily harm until it is triggered. The trigger releases the DNA from that of the host and employs it to create new viruses. [ citation needed ]


The genetic material in  retroviruses  is in the form of  RNA  molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and  integrase , into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed  reverse transcription . It is carried out by one of the enzymes carried in the virus, called  reverse transcriptase . After this DNA copy is produced and is free in the  nucleus  of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called  integrase . [ citation needed ]


Now that the genetic material of the virus has been inserted, it can be said that the host cell has been modified to contain new genes. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately. [ citation needed ]


One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus into any arbitrary position in the genome of the host; it randomly inserts the genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted ( insertional mutagenesis ). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e.,  cancer ) can occur. This problem has recently begun to be addressed by utilizing  zinc finger nucleases [ 1 ]  or by including certain sequences such as the  beta-globin locus control region  to direct the site of integration to specific chromosomal sites.


Gene therapy trials using retroviral vectors to treat X-linked  severe combined immunodeficiency  (X-SCID) represent the most successful application of gene therapy to date. More than twenty patients have been treated in France and Britain, with a high rate of immune system reconstitution observed. Similar trials were restricted or halted in the US when  leukemia  was reported in patients treated in the French X-SCID gene therapy trial. [ 2 ]  To date, four children in the French trial and one in the British trial have developed leukemia as a result of insertional mutagenesis by the retroviral vector. All but one of these children responded well to conventional anti-leukemia treatment. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase ( ADA ) enzyme (one form of SCID) [ 3 ]  continue with relative success in the US, Britain, Ireland, Italy and Japan. [ citation needed ]


Adenoviruses  are viruses that carry their genetic material in the form of double-stranded DNA. They cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are  transcribed  just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. [ citation needed ]


As a result, treatment with the adenovirus will require re-administration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has been promoted for treating cancer and indeed the first gene therapy product to be licensed to treat cancer,  Gendicine , is an adenovirus.  Gendicine, an adenoviral  p53-based  gene therapy was approved by the Chinese food and drug regulators in 2003 for treatment of head and neck cancer. Advexin, a similar gene therapy approach from Introgen, was turned down by the US  Food and Drug Administration  (FDA) in 2008.  [ 4 ]


Concerns about the safety of adenovirus vectors were raised after the 1999 death of  Jesse Gelsinger  while participating in a gene therapy trial. Since then, work using adenovirus vectors has focused on genetically limited versions of the virus. [ citation needed ]


Cytomegalovirus (CMV) is part of the β-herpesvirus subfamily that includes roseoloviruses. CMV coevolved with an assortment of mammalian hosts, including human CMV (HCMV), murine CMV (MCMV) and rhesus CMV (RhCMV). CMVs are characterized by large DNA genomes and typically asymptomatic infection in healthy hosts.


The first investigation into cytomegalovirus (CMV) as a gene therapy vector was published in 2000. CMV's tropism for hematopoietic progenitor cells and its large genome (230 kbp) initially attracted researchers. [ 5 ]  CMV-based vaccine vectors have since been used to induce T Cell response. [ 6 ]  More recently, CMV containing telomerase and follistatin was intravenously and intranasally delivered in mouse studies with the intention of extending healthspan. [ 7 ]


The viral vectors described above have natural host cell populations that they infect most efficiently.  Retroviruses  have limited natural host cell ranges, and although  adenovirus  and  adeno-associated virus  are able to infect a relatively broader range of cells efficiently, some cell types are resistant to infection by these viruses as well. Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a single protein coating their membrane, while adenoviruses are coated with both an envelope protein and fibers that extend away from the surface of the virus. The  envelope proteins  on each of these viruses bind to  cell-surface molecules  such as  heparin sulfate , which localizes them upon the surface of the potential host, as well as with the specific  protein receptor  that either induces entry-promoting structural changes in the viral protein, or localizes the virus in  endosomes  wherein acidification of the  lumen  induces this refolding of the  viral coat . In either case, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the cell. [ citation needed ]


For the purposes of gene therapy, one might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins. Such  chimera  would consist of those parts of the viral protein necessary for incorporation into the virion as well as sequences meant to interact with specific host cell proteins. Viruses in which the envelope proteins have been replaced as described are referred to as  pseudotyped viruses . For example, the most popular retroviral vector for use in gene therapy trials has been the  lentivirus   Simian immunodeficiency virus  coated with the envelope proteins,  G-protein , from  Vesicular stomatitis virus . This vector is referred to as  VSV G-pseudotyped lentivirus , and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one or a few host cell populations. This advance would allow for the systemic administration of a relatively small amount of vector. The potential for off-target cell modification would be limited, and many concerns from the medical community would be alleviated. Most attempts to limit tropism have used chimeric envelope proteins bearing  antibody  fragments. These vectors show great promise for the development of "magic bullet" gene therapies. [ citation needed ]


A replication-competent vector called ONYX-015 is used in replicating tumor cells. It was found that in the absence of the E1B-55Kd viral protein, adenovirus caused very rapid apoptosis of infected, p53(+) cells, and this results in dramatically reduced virus progeny and no subsequent spread. Apoptosis was mainly the result of the ability of EIA to inactivate p300. In p53(-) cells, deletion of E1B 55kd has no consequence in terms of apoptosis, and viral replication is similar to that of wild-type virus, resulting in massive killing of cells. [ citation needed ]


A replication-defective vector deletes some essential genes. These deleted genes are still necessary in the body so they are replaced with either a helper virus or a DNA molecule. [ 8 ]


Replication-defective vectors always contain a "transfer construct". The transfer construct carries the gene to be transduced or "transgene". The transfer construct also carries the sequences which are necessary for the general functioning of the viral genome: packaging sequence, repeats for replication and, when needed, priming of reverse transcription. These are denominated cis-acting elements, because they need to be on the same piece of DNA as the viral genome and the gene of interest. Trans-acting elements are viral elements, which can be encoded on a different DNA molecule. For example, the viral structural proteins can be expressed from a different genetic element than the viral genome. [ 8 ]


The  herpes simplex virus  is a human neurotropic virus.  This is mostly examined for gene transfer in the nervous system.  The wild type HSV-1 virus is able to infect neurons and evade the host immune response, but may still become reactivated and produce a lytic cycle of viral replication.  Therefore, it is typical to use mutant strains of HSV-1 that are deficient in their ability to replicate.  Though the latent virus is not transcriptionally apparent, it does possess neuron specific promoters that can continue to function normally. [ further explanation needed ]  Antibodies to HSV-1 are common in humans, however complications due to herpes infection are somewhat rare. [ 9 ]   Caution for rare cases of encephalitis must be taken and this provides some rationale to using HSV-2 as a viral vector as it generally has tropism for neuronal cells innervating the urogenital area of the body and could then spare the host of severe pathology in the brain. [ citation needed ]


Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of  transfection  and  expression of the gene  held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses. [ 10 ]


This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a  naked DNA  plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked  PCR  product, which have had similar or greater success. Cellular uptake of naked DNA is generally inefficient. Research efforts focusing on improving the efficiency of naked DNA uptake have yielded several novel methods, such as  electroporation ,  sonoporation , and the use of a " gene gun ", which shoots DNA coated gold particles into the cell using high pressure gas. [ 11 ]


Electroporation  is a method that uses short pulses of high voltage to carry DNA across the cell membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through. Electroporation is generally efficient and works across a broad range of cell types. However, a high rate of cell death following electroporation has limited its use, including clinical applications.


More recently a newer method of electroporation, termed electron-avalanche transfection, has been used in gene therapy experiments. By using a high-voltage plasma discharge, DNA was efficiently delivered following very short (microsecond) pulses. Compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage.


The use of particle bombardment, or the  gene gun , is another physical method of DNA transfection. In this technique, DNA is coated onto gold particles and loaded into a device which generates a force to achieve penetration of the DNA into the cells, leaving the gold behind on a "stopping" disk.


Sonoporation  uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to move into cells.


In a method termed  magnetofection , DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.


Hydrodynamic delivery  involves rapid injection of a high volume of a solution into vasculature (such as into the  inferior vena cava ,  bile duct , or  tail vein ). The solution contains molecules that are to be inserted into cells, such as  DNA plasmids  or  siRNA , and transfer of these molecules into cells is assisted by the elevated hydrostatic pressure caused by the high volume of injected solution. [ 12 ] [ 13 ] [ 14 ]


The use of synthetic oligonucleotides in gene therapy is to deactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses  antisense  specific to the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA called  siRNA  to signal the cell to cleave specific unique sequences in the  mRNA  transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene. The oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair. This technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted nucleotide alteration.


To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and positively charged. Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, in spite of the facts that there is little toxicity associated with them, that they are compatible with body fluids and that there was a possibility of adapting them to be tissue specific; they are complicated and time-consuming to produce so attention was turned to the cationic versions.


Cationic lipids , due to their positive charge, were first used to condense negatively charged DNA molecules so as to facilitate the encapsulation of DNA into liposomes. Later it was found that the use of cationic lipids significantly enhanced the stability of lipoplexes. Also as a result of their charge, cationic liposomes interact with the cell membrane,  endocytosis  was widely believed as the major route by which cells uptake lipoplexes. Endosomes are formed as the results of endocytosis, however, if genes can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to lysosomes where all DNA will be destroyed before they could achieve their functions. It was also found that although cationic lipids themselves could condense and encapsulate DNA into liposomes, the transfection efficiency is very low due to the lack of ability in terms of "endosomal escaping". However, when helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much higher transfection efficiency was observed. Later on, it was discovered that certain lipids have the ability to destabilize endosomal membranes so as to facilitate the escape of DNA from endosome, therefore those lipids are called fusogenic lipids. Although cationic liposomes have been widely used as an alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which could limit their therapeutic usages. [ 15 ]


The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Recent studies have shown lipoplexes to be useful in transfecting respiratory  epithelial cells .


Polymersomes  are synthetic versions of  liposomes  ( vesicles  with a  lipid bilayer ), made of  amphiphilic   block copolymers . They can encapsulate either  hydrophilic  or  hydrophobic  contents and can be used to deliver cargo such as DNA, proteins, or drugs to cells. Advantages of polymersomes over liposomes include greater stability, mechanical strength, blood circulation time, and storage capacity. [ 16 ] [ 17 ] [ 18 ]


Complexes of polymers with DNA are called polyplexes. [ 15 ] [ 19 ]  Most polyplexes consist of cationic polymers and their fabrication is based on self-assembly by ionic interactions. One important difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot directly release their DNA load into the cytoplasm. As a result, co-transfection with endosome-lytic agents such as inactivated adenovirus was required to facilitate nanoparticle escape from the endocytic vesicle made during particle uptake. However, a better understanding of the mechanisms by which DNA can escape from endolysosomal pathway, i.e. proton sponge effect, [ 20 ]  has triggered new polymer synthesis strategies such as incorporation of protonable residues in polymer backbone and has revitalized research on polycation-based systems. [ 21 ]


Due to their low toxicity, high loading capacity, and ease of fabrication, polycationic nanocarriers demonstrate great promise compared to their rivals such as viral vectors which show high immunogenicity and potential carcinogenicity, and lipid-based vectors which cause dose dependence toxicity.  Polyethyleneimine [ 22 ]  and  chitosan  are among the polymeric carriers that have been extensively studied for development of gene delivery therapeutics. Other polycationic carriers such as poly(beta-amino esters) [ 23 ]  and polyphosphoramidate [ 24 ]  are being added to the library of potential gene carriers. In addition to the variety of polymers and copolymers, the ease of controlling the size, shape, surface chemistry of these polymeric nano-carriers gives them an edge in targeting capability and taking advantage of enhanced permeability and retention effect. [ 25 ]


A  dendrimer  is a highly branched  macromolecule  with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface.


In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material such as DNA or RNA, charge complementarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis.


In recent years the benchmark for transfection agents has been cationic lipids. Limitations of these competing reagents have been reported to include: the lack of ability to transfect some cell types, the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. Dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size. Together these give compelling advantages compared to existing approaches.


Producing dendrimers has historically been a slow and expensive process consisting of numerous slow reactions, an obstacle that severely curtailed their commercial development. The Michigan-based company Dendritic Nanotechnologies discovered a method to produce dendrimers using kinetically driven chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from over a month to several days. These new "Priostar" dendrimers can be specifically constructed to carry a DNA or RNA payload that transfects cells at a high efficiency with little or no toxicity. [ citation needed ]


Inorganic nanoparticles, such as  gold ,  silica , iron oxide (ex.  magnetofection ) and  calcium phosphates  have been shown to be capable of gene delivery. [ 26 ]  Some of the benefits of inorganic vectors is in their storage stability, low manufacturing cost and often time, low immunogenicity, and resistance to microbial attack. Nanosized materials less than 100 nm have been shown to efficiently trap the  DNA  or  RNA  and allows its escape from the  endosome  without degradation. Inorganics have also been shown to exhibit improved in vitro  transfection  for attached cell lines due to their increased density and preferential location on the base of the culture dish.  Quantum dots  have also been used successfully and permits the coupling of gene therapy with a stable fluorescence marker. Engineered organic nanoparticles are also under development, which could be used for co-delivery of genes and therapeutic agents. [ 27 ]


Cell-penetrating peptides  (CPPs), also known as peptide transduction domains (PTDs), are short  peptides  (< 40 amino acids) that efficiently pass through cell membranes while being covalently or non-covalently bound to various molecules, thus facilitating these molecules' entry into cells. Cell entry occurs primarily by  endocytosis  but other entry mechanisms also exist. Examples of cargo molecules of CPPs include  nucleic acids ,  liposomes , and drugs of low molecular weight. [ 28 ] [ 29 ]


CPP cargo can be directed into specific cell organelles by incorporating  localization sequences  into CPP sequences. For example,  nuclear localization sequences  are commonly used to guide CPP cargo into the nucleus. [ 30 ]  For guidance into mitochondria, a  mitochondrial targeting sequence  can be used; this method is used in  protofection  (a technique that allows for foreign  mitochondrial DNA  to be inserted into cells' mitochondria). [ 31 ] [ 32 ]


Due to every method of  gene transfer  having shortcomings, there have been some hybrid methods developed that combine two or more techniques.  Virosomes  are one example; they combine  liposomes  with an inactivated  HIV  or  influenza virus . This has been shown to have more efficient gene transfer in respiratory  epithelial cells  than either viral or liposomal methods alone. Other methods involve mixing other viral vectors with  cationic lipids  or  hybridising viruses . [ citation needed ]


Ecogenetics  is a branch of  genetics  that studies  genetic traits  related to  the response to environmental substances . [ 1 ]  Or, a contraction of  ecological genetics , the study of the relationship between a natural population and its genetic structure. [ citation needed ]


Ecogenetics principally deals with effects of preexisting genetically-determined variability on the response to environmental agents. [ 2 ]  The word environmental is defined broadly to include the physical, chemical, biological, atmospheric, and climate agents. Ecogenetics, therefore, is an all-embracing term, and concepts such as pharmacogenetics are seen as subcomponents of ecogenetics. This work grew logically from the book entitled  Pollutants and High Risk Groups  (1978), which presented an overview of the various host factors i.e. age, heredity, diet, preexisting diseases, and lifestyles which affect environmentally-induced disease.


The primary intention of ecogenetics is to provide an objective and critical evaluation of the scientific literature pertaining to genetic factors and differential susceptibility to environmental agents, with particular emphasis on those agents typically considered pollutants. It is important to realize though that ones genetic makeup, while important, is but one of an array of host factors contributing to overall adaptive capacity of the individual. In many instances, it is possible for such factors to interact in ways that may enhance or offset the effect of each other.


Red blood cell conditions
There is a broad group of genetic diseases that result in either producing or predisposing affected individuals to the development of hemolytic anemias. These diseases include abnormal haemoglobin, inability to manufacture one or the other of the peptide globin chains of the haemoglobin, and deficiencies of the Embden-Meyerhoff monophosphate.


Liver metabolism
Individuals lacking the ability to detoxify and excrete PCB's may have a high risk of total liver failure in conjunction with certain ecological conditions.


Cardiovascular diseases
The pathologic lesion of atherosclerosis is a plaque-like substance that thickens the innermost and middle of the three layers of the artery wall. The thickening of the intimal and medial layers results from the accumulation of the proliferating smooth muscle cells that are encompassed by interstitial substances such as  collagen ,  elastin ,  glycosaminoglycans , and  fibrin .


Respiratory diseases
There are three genetically-based respiratory diseases that can directly correspond with ecological functions and induce disease. These include lung cancer and the upper and lower respiratory tract associated with a serum Ig A deficiency.


This template is used to identify a  genetics  stub. It uses {{ asbox }}, which is a meta-template designed to ease the process of creating and maintaining stub templates.


Typing  {{Genetics-stub}}  produces the message shown at the beginning, and adds the article to the following category:


This is a  stub template . A brief explanation of these templates follows; for full details please consult  Wikipedia:Stub .


A stub is an article containing only a few sentences of text which is too short to provide encyclopedic coverage of a subject.


Further information can be found at:


New stub templates and categories (collectively "stub types") should not be created without prior proposal at  Wikipedia:WikiProject Stub sorting/Proposals . This allows for the proper coordination of all stub types across Wikipedia, and for the checking of any new stub type for possible problems prior to its creation.


This template is used to identify a stub about a non-fiction book on  genetics . It uses {{ asbox }}, which is a meta-template designed to ease the process of creating and maintaining stub templates.


Typing  {{Genetics-book-stub}}  produces the message shown at the beginning, and adds the article to the following categories:


The  A-site  (A for aminoacyl) of a  ribosome  is a  binding site  for charged  t-RNA  molecules during  protein synthesis . [ 1 ]   One of three such binding sites, the A-site is the first location the t-RNA binds during the protein synthesis process, the other two sites being  P-site  (peptidyl) and  E-site  (exit).


An  active chromatin sequence  (ACS) is a region of  DNA  in a  eukaryotic   chromosome  in which  histone  modifications such as  acetylation  lead to exposure of the  DNA sequence  thus allowing binding of  transcription factors  and  transcription  to take place. Active chromatin may also be called  euchromatin . ACSs may occur in non-expressed gene regions which are assumed to be "poised" for  transcription . The sequence once exposed often contains a promoter to begin transcription. At this site  acetylation  or  methylation  can take place causing a conformational change to the chromatin. At the active chromatin sequence site deacetylation can cause the gene to be repressed if not being expressed.


Chromatin


Adam's Curse: A Future Without Men  (also known as  Adam's Curse: A Story of Sex, Genetics, and the Extinction of Men ) is a 2003 book by  Oxford University  human  genetics  professor  Bryan Sykes  expounding his hypothesis that with the declining  sperm count  in men and the continual atrophy of the  Y chromosome , within 5,000 generations (approximately 125,000 years) men shall become extinct.


Sykes thinks one of the options for humanity's survival is unisex reproduction by females: female eggs fertilised by the nuclear  X chromosomes  of another female and implanted using  in vitro fertilisation  methods. He also introduces the possibility of moving the  SRY  and associated genes responsible for maleness and male fertility to another chromosome, which he refers to as "the Adonis chromosome", engendering fertile males with an XX  karyotype .


BBC News reported in 2012 that a US study in Nature suggests the genetic decay has all but ended and that "The conclusion from these comparative studies is that genetic decay has in recent history been minimal, with the human chromosome having lost no further genes in the last six million years, and only one in the last 25 million years." [ 1 ]


This article about a book on  futures studies  or  futurology  is a  stub . You can help Wikipedia by  expanding it .


An  adapter  or adaptor in  genetic engineering  is a short, chemically synthesized, double-stranded  oligonucleotide  that can be ligated to the ends of other DNA or RNA molecules. Double stranded adapters are different from linkers in that they contain one blunt end and one sticky end.  [ 1 ]  For instance, a  double stranded   DNA  adapter can be used to link the  ends  of two other DNA molecules (i.e., ends that do not have "sticky ends", that is complementary protruding single strands by themselves). It may be used to add  sticky ends  to  cDNA  allowing it to be ligated into the  plasmid  much more efficiently. [ 2 ]  Two adapters could base pair to each other to form  dimers .


A conversion adapter is used to join a DNA insert cut with one  restriction enzyme ,  say  EcoRl , with a vector opened with another enzyme,  Bam Hl . This adapter can be used to convert the cohesive end produced by Bam Hl to one produced by Eco Rl or vice versa.
One of its applications is ligating cDNA into a plasmid [ 3 ]  or other vectors instead of using Terminal deoxynucleotide Transferase enzyme to add poly A to the cDNA fragment. [ 4 ]


NGS adapters are short ~80 BP fragments that bind to DNA to aid in amplification during library preparation and are also useful to bind DNA to the flow cell during sequencing.  [ 5 ]  These adapters are made up of three parts that flank the DNA sequence of interest. There is the flow cell binding sequence, the primer binding site, and also tagged barcoded regions to allow pooled sequencing.


Genetic effects are broadly divided into two categories: additive and non-additive.  Additive genetic effects  occur where expression of more than one gene contributes to  phenotype  (or where  alleles  of a  heterozygous  gene both contribute), and the phenotypic expression of these gene(s) can be said to be the sum of these contributions.


Non-additive effects involve  dominance  or  epistasis , and cause outcomes that are not a sum of the contribution of the genes involved.


Additive genetic effects are singularly important with regard to  quantitative traits , as the sum of these effects informs the placement of a trait on the spectrum of possible outcomes. Quantitative traits are commonly polygenic (resulting from the effects of more than one  locus ).


Variation in phenotypes across a population arises from the interaction between environmental variation and genetic variation between individuals. This can be stated mathematically as:


V P  = V E  + V G , where the terms refer to variation in phenotype, environment, and genotype respectively. [ 1 ]


Broad sense heritability (H 2 , or H B ) refers to the phenotypic differences arising from all genetic effects, and can be described as the ratio of genotypic variation to that of phenotypic variation in the population, or: H 2  = V G  / V P .


The genotypic variation from the above equation can be subdivided into V A  (additive effects), V D  (dominance effects), and V I  (epistatic effects).


Narrow sense Heritability (h 2  or H N ) focuses specifically on the ratio of additive variance (V A ) to total phenotypic variance (V P ), or: h 2  = V A  / V P.


In the study of Heritability, Additive genetic effects are of particular interest in the fields of  Conservation , and  Artificial selection . The effects of dominance and epistasis are not reliably transmitted to progeny (see  Mendelian inheritance , laws of segregation and independent assortment). This means that h 2  represents the phenotypic variation that is reliably passed from one generation to the next and which can be used to predict changes in mean fitness between generations.  [ 2 ]


Fisher's Fundamental Theorem  asserts a direct correlation between the amount of genetic variation in a population and the possible variation in mean fitness for that population. This Theorem suggests that where a trait affects individual fitness, the amount of variation due to additive genetic effects will decline with each successive generation, and all other things being equal, will approach zero.


The alternative flatworm mitochondrial code  (translation table 14) is a  genetic code  found in the mitochondria of  Platyhelminthes   and  Nematodes .


Bases:  adenine  (A),  cytosine  (C),  guanine  (G) and  thymine  (T) or  uracil  (U).


Amino acids:  Alanine  (Ala, A),  Arginine  (Arg, R),  Asparagine  (Asn, N),  Aspartic acid  (Asp, D),  Cysteine  (Cys, C),  Glutamic acid  (Glu, E),  Glutamine  (Gln, Q),  Glycine  (Gly, G),  Histidine  (His, H),  Isoleucine  (Ile, I),  Leucine  (Leu, L),  Lysine  (Lys, K),  Methionine  (Met, M),  Phenylalanine  (Phe, F),  Proline  (Pro, P),  Serine  (Ser, S),  Threonine  (Thr, T),  Tryptophan  (Trp, W),  Tyrosine  (Tyr, Y),  Valine  (Val, V)


Code 14 differs from code 9 ( the echinoderm and flatworm mitochondrial code ) only by translating UAA to Tyr rather than STOP.  A study in 2000 [ 1 ]  has found no evidence that the codon UAA codes for Tyr in the flatworms but other opinions exist.  There are very few  GenBank  records that are translated with code 14 but a test translation shows that re-translating these records with code 9 can cause premature terminations.  More recently, UAA has been found to code for tyrosine in the nematodes  Radopholus similis [ 2 ]  and  Radopholus arabocoffeae . [ 3 ]


This article incorporates text from the  United States National Library of Medicine , which is in the  public domain .
 [ 4 ]


The  alternative yeast nuclear code  (translation table  12 ) is a  genetic code  found in certain yeasts.  However, other yeast, including  Saccharomyces cerevisiae ,  Candida azyma ,  Candida diversa ,  Candida magnoliae ,  Candida rugopelliculosa ,  Yarrowia lipolytica , and  Zygoascus hellenicus , definitely use the standard (nuclear) code. [ 1 ]


Amino acids:  Alanine  (Ala, A),  Arginine  (Arg, R),  Asparagine  (Asn, N),  Aspartic acid  (Asp, D),  Cysteine  (Cys, C),  Glutamic acid  (Glu, E),  Glutamine  (Gln, Q),  Glycine  (Gly, G),  Histidine  (His, H),  Isoleucine  (Ile, I),  Leucine  (Leu, L),  Lysine  (Lys, K),  Methionine  (Met, M),  Phenylalanine  (Phe, F),  Proline  (Pro, P),  Serine  (Ser, S),  Threonine  (Thr, T),  Tryptophan  (Trp, W),  Tyrosine  (Tyr, Y),  Valine  (Val, V).


This article incorporates text from the  United States National Library of Medicine , which is in the  public domain .
 [ 3 ]


Ambidirectional dominance  occurs in a situation where multiple  genes  influence a  phenotype  and  dominance  is in different directions depending on the gene. For example, for  gene  A increased height is dominant, while for  gene  B decreased height is dominant. The opposite situation, where all genes show dominance in the same direction, is called  directional dominance . [ 1 ]  In the same example, for both genes A and B increased height is dominant. According to Broadhurst, ambidirectional dominance is the result of  stabilising selection  in the evolutionary past. [ 1 ]  Ambidirectional dominance has been found for  exploratory behaviours  in  mice [ 2 ] [ 3 ] [ 4 ]  and  paradise fish . [ 5 ]


American Society of Gene & Cell Therapy  (ASGCT) is a professional  non-profit  medical and scientific organization based in  Milwaukee , dedicated to understanding, development and application of  gene , related  cell  and  nucleic acid  therapies, as well as promotion of professional and public education on gene therapy. With more than 4,800 members in the United States and worldwide, today ASGCT is the largest association of individuals involved in gene and cell therapy research. [ 1 ]


Molecular Therapy  is the official journal of the ASGCT.


The  Animal Genome Size Database  is a catalogue of published  genome size  estimates for  vertebrate  and  invertebrate   animals . [ 1 ]   It was created in 2001 by  Dr. T. Ryan Gregory  of the  University of Guelph  in Canada.  As of September 2005, the database contains data for over 4,000 species of animals.  A similar  database , the  Plant DNA C-values Database  ( C-value  being analogous to  genome size  in  diploid   organisms ) was created by researchers at the  Royal Botanic Gardens, Kew , in 1997.


Artificially Expanded Genetic Information System  ( AEGIS ) is a  synthetic DNA analog  experiment that uses some  unnatural base pairs  from the laboratories of the  Foundation for Applied Molecular Evolution  in  Gainesville, Florida . AEGIS is a  NASA -funded project to try to understand how  extraterrestrial life  may have developed. [ 1 ]


The system uses twelve different  nucleobases  in its  genetic code . These include the four canonical nucleobases found in  DNA  ( adenine ,  cytosine ,  guanine  and  thymine ) plus eight  synthetic nucleobases  ( S ,  B ,  Z ,  P , V, J,  K , and X). [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ]  AEGIS includes S:B, Z:P, V:J and K:X base pairs. [ 6 ] [ 7 ]


This  astrobiology -related article is a  stub . You can help Wikipedia by  expanding it .


The ascidian mitochondrial code  (translation table  13 ) is a  genetic code  found in the mitochondria of  Ascidia .


There is evidence from a phylogenetically diverse sample of  tunicates  ( Urochordata ) that AGA and AGG code for  glycine .  In other organisms, AGA/AGG code for either  arginine  or  serine  and in vertebrate  mitochondria  they code a STOP.  Evidence for glycine translation of AGA/AGG was first found in 1993 in  Pyura stolonifera [ 1 ]  and  Halocynthia roretzi . [ 2 ]  It was then confirmed by tRNA sequencing [ 3 ]  and sequencing whole mitochondrial genomes. [ 4 ] [ 5 ]


This article incorporates text from the  United States National Library of Medicine , which is in the  public domain .
 [ 7 ]


Atypical Solute Carrier Families  (Atypical SLCs) are novel plausible secondary active or facilitative transporter proteins that share ancestral background with the known  solute carrier families (SLCs) . However, they have not been assigned a name according to the SLC root system, or been classified into any of the existing SLC families. [ 1 ] [ 2 ]


Most atypical SLCs are  families  within the  major facilitator superfamily  (MFS). [ 3 ]  These atypical SLCs are plausible secondary active or facilitative transporter proteins that share ancestry with the known  solute carriers . [ 1 ] [ 2 ] [ 4 ]  They are, however, not named according to the SLC root system, or classified into any of the existing SLC families. [ 1 ]  ATMFs are categorised based on their sequence similarity and phylogenetic closeness. [ 3 ]


Some Atypical SLC of MFS type are:  OCA2 ,  CLN3 , SPNS1,  SPNS2 , SPNS3,  SV2A ,  SV2B , SV2C, SVOP, SVOPL,  MFSD1 ,  MFSD2A , MFSD2B,  MFSD3 , MFSD4A, [ 5 ]  MFSD4B,  MFSD5 , MFSD6, MFSD6L,  MFSD8 ,  MFSD9 , [ 5 ]  MFSD10,  MFSD11 , MFSD12, MFSD13A,  MFSD14A ,  MFSD14B ,  UNC93A [ 6 ]  and  UNC93B1 . All these are atypical SLCs found within the  Major facilitator superfamily . Also TMEM104 (APC clan), OCA2 (IT clan) and CLN3 (having no clan) are atypical SLCs in humans.  [ 7 ] [ 8 ] [ 9 ]


Although most atypical SLCs are from the major facilitator superfamily, there are exceptions: TMEM104 ( APC superfamily ), OCA2 (IT superfamily) and CLN3 (unknown superfamily). [ 1 ]


The  B recognition element  ( BRE ) is a  DNA sequence  found in the  promoter region  of most [ clarification needed ]  genes in  eukaryotes  and  Archaea . [ 1 ] [ 2 ]   The BRE is a  cis-regulatory element  that is found immediately near  TATA box , and consists of 7  nucleotides . There are two sets of BREs: one (BREu) found immediately upstream of the TATA box, with the consensus SSRCGCC; the other (BREd) found around 7 nucleotides downstream, with the consensus RTDKKKK. [ a ] [ 3 ] [ 4 ]


The BREu was discovered in 1998 by  Richard Ebright  and co-workers. [ 1 ]  The BREd was named in 2005 by Deng and Roberts; [ 5 ]  such a downstream recognition was reported earlier in 2000 in Tsai and Sigler's crystal structure. [ 6 ]


The  transcription factor II B  (TFIIB) recognizes either BRE and binds to it. Both BREs work in conjunction with the TATA box (and  TATA box binding protein ), and have various effects on levels of transcription. [ 4 ]


TFIIB uses the  cyclin -like repeats to recognize DNA. The C-terminal  alpha helices  of TFIIB intercalate with the  major groove  of the DNA at the BREu. The N-terminal helices bind to the minor groove at BREd. TFIIB is one part of the  preinitiation complex  that helps  RNA polymerase II  bind to the DNA. [ 6 ]


In addition to the human TFIIB-BRE structure, structures from many other organisms have been solved. Among those are  transcription factor B  (TFB) from the  archaeon   Pyrococcus woesei  which presents an inverted orientation [ 7 ]  and a TFIIB from the parasite  Trypanosoma brucei  which despite some specific insertions show a similar fold. [ 8 ]


B-cell CLL/lymphoma  refers to a family of genes associated with certain types of  lymphoma  and  leukemia .


Although named for  B-cell chronic lymphocytic leukemia , they can be associated with other malignancies.


Members include:


In  population genetics , the  Balding–Nichols model  is a statistical description of the  allele frequencies  in the components of a sub-divided population. [ 1 ]  With background allele frequency  p  the allele frequencies, in sub-populations separated by  Wright's   F ST   F , are distributed according to independent draws from


where  B  is the  Beta distribution . This distribution has mean  p  and variance  Fp (1 –  p ). [ 2 ]


The model is due to  David Balding  and  Richard Nichols  and is widely used in the forensic analysis of  DNA profiles  and in population models for  genetic epidemiology .


Basic helix-loop-helix leucine zipper transcription factors  are, as their name indicates,  transcription factors  containing both  Basic helix-loop-helix  and  leucine zipper  motifs.


Examples include  Microphthalmia-associated transcription factor  and  Sterol regulatory element binding protein  (SREBP).


The  bead theory  is a disproved  hypothesis  that  genes  are arranged on the  chromosome  like  beads  on a  necklace . This theory was first proposed by  Thomas Hunt Morgan  after discovering  genes  through his work with breeding red and white eyed fruit flies. [ 1 ]  According to this theory, the existence of a gene as a unit of inheritance is recognized through its mutant alleles. A mutant allele affects a single phenotypic character, maps to one chromosome locus, gives a mutant phenotype when paired and shows a Mendelian ratio when intercrossed. Several tenets of the bead theory are worth emphasizing :-
1. The gene is viewed as a  fundamental unit of structure , indivisible by crossing over. Crossing over take place between genes ( the beads in this model ) but never within them.
2. The gene is viewed as the  fundamental unit of change or mutation . It changes in toto from one allelic form into another; there are no smaller components within it that can change.
3. The gene is viewed as the  fundamental unit of function  ( although the precise function of gene is not specified in this model ). Parts of a gene, if they exist cannot function.  Guido Pontecorvo  continued to work under the basis of this theory until
 Seymour Benzer  showed in the 1950s that the bead theory was not correct. [ 2 ]  He demonstrated that a gene can be defined as a unit of function. A gene can be subdivided into a linear array of sites that are mutable and that can be recombined. The smallest units of mutation and recombination are now known to be correlated with single nucleotide pairs.


New york W.H. Freeman;2000


1EFH ,  1J99 ,  1OV4 ,  2QP3 ,  2QP4 ,  3F3Y ,  4IFB


Bile salt sulfotransferase  also known as   hydroxysteroid sulfotransferase  (HST) or  sulfotransferase 2A1  (ST2A1) is an  enzyme  that in humans is encoded by the  SULT2A1   gene . [ 5 ] [ 6 ] [ 7 ]


Sulfotransferase  enzymes catalyze the  sulfate conjugation  of many hormones, neurotransmitters, drugs, and xenobiotic compounds. These cytosolic enzymes are different in their tissue distributions and substrate specificities. The gene structure (number and length of exons) is similar among family members. This gene is primarily expressed in liver and adrenal tissues where the encoded protein sulfonates steroids and bile acids. [ 5 ]


This  transferase  article is a  stub . You can help Wikipedia by  expanding it .


Biostrophin  is a drug which may serve as a vehicle for  gene therapy , in the treatment of  Duchenne  and  Becker   muscular dystrophy . [ 1 ]


As  mutations  in the  gene  which codes for the protein  dystrophin  is the underlying defect responsible for both
disorders, biostrophin will deliver a genetically-engineered, functional copy of the gene at the  molecular  level to affected muscle cells. [ 1 ]  Dosage, as well as a viable means for systemic release of the drug in patients, is currently being investigated with the use of both  canine  and  primate   animal models . [ 2 ]


Biostrophin is being manufactured by Asklepios BioPharmaceuticals, Inc., with funding provided by the  Muscular Dystrophy Association . [ 3 ]


This  drug  article relating to the  musculoskeletal system  is a  stub . You can help Wikipedia by  expanding it .


BMN 331  is an experimental  gene therapy  for  hereditary angioedema , delivered by  adeno-associated virus  type 5 and targeting the  SERPING1  gene. [ 1 ] [ 2 ]


In  agriculture  and in  animal fancy , a  breeder  is an animal used for  selective breeding . A breeder is usually a  purebred  animal, bred with the intent of producing purebred, or even show-quality animals. However, in some cases, a breeding animal is  crossbred  with another breed or a  mixed breed  with the intent of combining aspects of two or more different breeds.


If the breeding is for a purebred animal that will be used for exhibition or future breeding ( pets  or  livestock ), the animal must be  registered  and conform to the criteria laid out for that  breed  in a  breed standard  kept by a central authority, such as a  kennel club  for dogs. In addition, the breed club, kennel club, or other governing authority may have other restrictions on the type of animal that can be used for breeding to produce offspring that can be registered.


For example, some horse registries allow  backbred  and  crossbred  individuals to be breeders. However, most dog registries do not accept crossbred animals for registration, except in exceptional circumstances and only by permission. Most kennel clubs allow any registered individual to be a breeder, however the individual breed club may have additional criteria such as having to pass certain eligibility requirements.


In animal fancy, there are specialized words for breeding animals in some species. (Not to be confused with the words for an animal's parents,  sire  and  dam .)


This  agriculture  article is a  stub . You can help Wikipedia by  expanding it .


The  Britten–Davidson  model, [ 1 ] [ 2 ]  also known as the gene-battery model, [ 3 ]  is a hypothesis for the regulation of  protein synthesis  in  eukaryotes . Proposed by  Roy John Britten  and  Eric H. Davidson  in 1969, the model postulates four classes of  DNA  sequence: an integrator gene, a producer gene, a receptor site, and a sensor site. The sensor site regulates the integrator gene, responsible for synthesis of activator RNA. The integrator gene cannot synthesize activator RNA unless the sensor site is activated. Activation and deactivation of the sensor site is done by external stimuli, such as  hormones . The activator RNA then binds with a nearby receptor site, which stimulates the synthesis of  mRNA  at the  structural gene .


This theory would explain how several different integrators could be concurrently synthesized, and would explain the pattern of  repetitive DNA  sequences followed by a unique  DNA  sequence that exists in  genes . [ 4 ]


The  Burkholderiales-2 RNA motif  is a conserved  RNA  structure that was  discovered by bioinformatics . [ 1 ]
Burkholderiales-2 motifs are found in  Betaproteobacteria . Although one example is predicted in the phylum  Bacteroidota , this is likely to be the result of a recent  horizontal gene transfer  or sequence contamination.


Burkholderiales-2 RNAs likely function in  trans  as  sRNAs .  There is weak evidence of an association with S24  peptidases .


Buttien-Fryns syndrome  is a congenital  genetic disorder  that causes severe  oligodactyly  and  micrognathia . It is caused by a change in the structure of the 10q  gene . [ 1 ]  The condition has been reported in four patients, two of which were siblings. [ 2 ]


Oligodactyly and micrognathia are the most well known symptoms of the disease. Other symptoms include: [ 3 ]


Buttien-Fryns syndrome is caused by a duplication or triplication of the 10q24 gene. [ 4 ]  This gene is also associated with other conditions such as  split hand . [ 1 ]  The condition is inherited in an autosomal recessive manner. [ 3 ]


In  molecular biology , and more importantly  high-throughput DNA sequencing , a  chimera  is a single DNA sequence originating when multiple  transcripts  or DNA sequences get joined. Chimeras can be considered  artifacts  and be filtered out from the data during processing  [ 1 ]  to prevent spurious inferences of biological variation. [ 2 ]  However, chimeras should not be confused with chimeric  reads , who are generally used by structural variant callers to detect  structural variation  events [ 3 ]  and are not always an indication of the presence of a chimeric transcript or gene.


In a different context, the deliberate creation of artificial chimeras can also be a useful tool in molecular biology. For example, in  protein engineering , "chimeragenesis" (forming chimeras between proteins that are encoded by homologous  cDNAs ) [ 4 ]  is one of the "two major techniques used to manipulate cDNA sequences". [ 4 ]  For gene fusions that occur through natural processes, see  chimeric genes  and  fusion genes .


A chimera can occur as a single  cDNA  sequence originating from two  transcripts . It is usually considered to be a contaminant in transcript and  expressed sequence tag  (which results in the moniker of  EST chimera ) databases. [ 5 ]  It is estimated that approximately 1% of all transcripts in the  National Center for Biotechnology Information 's Unigene database contain a "chimeric sequence". [ 6 ]


A chimera can also be an artifact of  PCR  amplification. It occurs when the extension of an  amplicon  is aborted, and the aborted product functions as a  primer  in the next PCR cycle. The aborted product  anneals  to the wrong template and continues to extend, thereby synthesizing a single sequence sourced from two different templates. [ 7 ]


PCR chimeras are an important issue to take into account during  metabarcoding , where DNA sequences from environmental samples are used to determine biodiversity. A chimera is a novel sequence that will most probably not match to any known organism. Hence, it might be interpreted as a new species thereby overinflating the diversity.