does repetitive sequence in the arm of the targeting vector affect recombination in gene targeting?
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Introduction. This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as "knock-out" mice or "gene targeted" mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact Thom Saunders with any questions or Meet the Staff.  The Transgenic Core prioritizes all requests for service on a "first-come, first-serve" basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received. \n2. Plan the experiment. What is the purpose of your experiment? Do you want to completely ablate gene expression in all tissues? Do you want to analyze tissue specific regulatory sequences? Do you want to produce a model for tissue-specific gene deletions? You will need to obtain or clone the structural gene that you want to mutate. The source of the DNA is important since published studies demonstrate genomic DNA isogenic to the embryonic stem cell line give higher gene targeting efficiencies in ES cells (see Key Gene Targeting Papers). While complete sequence information is not necessary, a detailed restriction map of the gene is an irreplaceable tool. During the planning stages the material transfer agreements for R1 ES cells and other reagents should be initiated so that they are ready in time. Once a genomic clone of the gene is in hand and restriction mapped, then it is time to... \n\n3. Clone and the Verify the Targeting Vector. The Core has cloning vectors for investigators to use in gene targeting research. They include pPNT, a basic targeting vector, pNZTK2, designed to insert the lacZ gene behind the promoter of the targeted gene, and pflox, which is designed to introduce loxP sites into the gene. A complete materials transfer agreement is required before they can be distributed. You will need to determine which 5' and 3' fragments of the gene will be used to construct the targeting vector. Generally speaking, a couple of Kb on either end should give a reasonable gene targeting frequency. You will also need to consider the methods and probes that you will use to identify ES cell clones that have undergone homologous recombination with the targeting vector. This means a Southern blot approach with probes outside of the targeting vector or a PCR based assay with primers outside of the targeting vector. Once the orientation of the fragments in the targeting vector are confirmed and the probes are tested then it is time to... \n\n4. Electroporate ES Cells and Select Recombinants. ES cells are a very sensitive reagent, and most often the weakest link in the process of producing gene targeted mice. Fastidious tissue culture technique is necessary to maintain the totipotent state of the cells during electroporation, selection, and expansion. In order to maximize successful outcomes, the Transgenic Core maintains ES cells and companion reagents that are quality tested by producing ES cell-mouse chimeras that transmit the ES cell genome to offspring. Investigators may choose to use the Core's Gene Targeting Service (we do all of the tissue culture work, you do all of the molecular biology), or to use space in the Core lab (rent space in our completely equipped laboratory for ES cell culture work), or to do th
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From what I have read, it appears that it is autosomal dominant and is not X-linked as the gene most likely responsible lies on chromosome 1.\nHope this helps
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I don't think double stranded probe is a good idea. You can isolate the mRNA, then synthesize its complementary strand by using RNApolymerase. This complementary strand can be used as probe.\n\nYou can also prepare synthetic polynucleotide deduced from the sequence which would be complementary to cDNA. Like the codon for Leu may be TTA, TTG, CTT, CTC, CTA, CTG. So the corresponding complementary DNA sequence can be AAT, AAC, GAA, GAG, GAT, GAC. Actually you have to design and synthesize many probes using each of these 3 nucleotide sets, then if the cDNA is mixed, the complementary sequence will bind..... making an effective probe.\n\nYou can go through An Introduction to Genetic Engineering By Nicholl.
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We use transgenic mice all the time.  Your question is a bit vague though.
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Not a stupid question at all. Mutation (as mentioned above) does occur, though of course it will only affect individual cells and their descendents, not all cells in the individual. There is also a process that is being increasingly studied called genomic imprinting or epigenetic inheritance. These are not DNA sequence changes, like you asked about, but seem to be related to your question. The wikipedia article is a decent introduction but there are other summaries available.
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You have to identify two sequences that are >6kb apart from one another that flank the sequence you are attempting to amplify. Better yet, you can get you a well understood cloning vector (plasmid) that already has primers develped for it that flank the polyrestriction site region (ie. puc19 [see link below], find the needed restriction enzyme sites that flank your desired 6kb sequence and clone the DNA into the vector. Be sure to check your sequence for any internal restriction sites. This may require you to do a partial digestion and recovery.
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http://www-sequence.stanford.edu/group/techdev/plasmid.html
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I'm pretty sure that its random because in meiosis there is crossing over. The chiasmata are where the chromosomes cross, which is done randomly. Since the codons are part of the DNA, they would be crossing at random too. The only exception that I can think of is when genes are located at least within 50 map units of eachother on the chromosomes, which makes it more likely that the genes will cross over together.\nHope this helps!
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All answers are correct. The primary aminoacid sequence is changed which may have implications for the structure and funtion of the protein
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Direct application of recombinant DNA technology in people has so far been difficult.  The best known therapy is treatment of SCID (severe combined immunodeficiencies), aka 'bubble-boy disease'.  15 of the 16 patients treated responded to the gene therapy.  However, three of the 15 developed a leukemia-like disease, of whom one died.  The first article below gives a thorough, albeit highly technical, look at gene therapy in people.\n\nMore generally, recombinant DNA technology has been extremely useful in developing drugs for a variety of diseases.  Remicade, Anakinra, and Enbrel are useful drugs for rheumatoid arthritis and Crohn's disease.  Rituxan is useful for certain lymphomas, a type of cancer.  There are many other examples, these are just a few.
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There are two main sources of genetic recombination. The first is through cross-over events during meiosis between homologous chromosomes. The second is through gene conversion on the same chromosome.\n\nRecombination events create new combinations of alleles, and therefore increase genetic diversity. Recombination also increases the effectiveness of natural selection by increasing the liklihood that advantageous mutations are kept while increasing the liklihood that deleterious mutations are eliminated from the population.
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a mutation can change the DNA but not the amino acid sequence because in order to get a protein there are several options for DNA. (for example) you can UAG as a protein and you can get that by ATG, CGC, AUC and more.  So that gives you a chance to get the same amino acid with a change in the DNA.
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Microsatellites are expansive repeats of two, three, four, or five nucleotides (ex: GCCGCCGCCGCC...). They occur when the DNA template "slips" and the DNA replication machinery reorients itself on the template incorrectly. For example, imagine that there are 25 "GCC" repeats, and suppose that the DNA replicating machinery is on repeat 4. The template "slips" and the DNA replicating machinery goes back to repeat 2. Why does it not know that it has done this? Because there are so many GCC that it doesn't recognize the mistake.  So it was replicating #4 GCC, but now it's replicating #2 GCC. That means that the final DNA strand being replicated will now have two additional GCC...and thus, an increase in the microsatellite region.
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Increasing evidence suggests nonrandom localization of DNA sequences in the cell nucleus. Changes in intranuclear gene location as a function of transcriptional activity has been established in several experimental systems
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1. Isolate the genome of organism on which the desired gene sits.\n2. Cut it with restriction enzyme with the desired recognition site.\n3. Perform polymerase chain reaction procedure with oligomer primers which have the suspected complimentary sequence with your gene\n4. Perform agarose gel electophoresis to isolate the multiplied sequences\n5. Separate the duplex structure of primer:DNA in alkaline medium.\n6. Perform Southern Blot Transfer (could be found in any molecular biology laboratory manual)\n7. Transform the target organism with the resulting gene fragments from the southern blot. (transformation procedure vary with the varying target organism)
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I believe you're talking about Long Terminal Repeat sequences IN retrotransposons IN retroviruses.\n\nThe basics (just in case): A transposon or "jumping gene" is a bit of DNA that moves around the genome with the aid of enzymes called transposases. Your plain old bread-and-butter transposons are actually cut from the DNA strand and lygated into another, often random, part of the genome.\n\nNow, retrotransposons are special because they aren't actually cut out...they're merely copied (using reverse transcriptase...which is RNA dependent DNA polymerase). After their copied, they can be inserted into other parts of the genome. You can deduct how multiple copies of a gene in a virus could be beneficial.\n\nTo your question (finally, eh?): Long Terminal Repeats (LTRs) are on each end of a retrotransposon. They have the same orientation at both ends. Their function? Simply to say "Hey! Look at my data! I'm a retrotransposon! Copy me! Paste me!" See?\n\nHere's what LTRs look like:\n\nGCGCATCC------------GCGCATCC (where ------- is replaced by a gene) Keep in mind that LTRs are much, much longer than this example.\n\nHere's what a retrotransposon with LTR's might look like:\n\nGCGCATCC(TAGCGCAATCG)GCGCATCC\n\nThat help? :D
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I'm not sure what you are asking because of the typos. But here goes an answer for what I think you are trying to ask.\n\nA point mutation involving the addition of a BASE early in the coding, The protein resulting from The gene will be entirely different from the point of addition, since the reading frame will be moved forward. \n\nExample (HYPOTHETICAL)\n\nNormal DNA\nATCGGTACCATT\n\nNormal mRNA\nUAGCCAUGGUAA  The reading frame would be UAG-CCA-UGG-UAA...\n\nmutated mRNA (a C added by mistake)\nUAGCCCAUGGUAA The reading frame would be UAG-CCC-AUG-GUA..\n\nThis can cause the protein to be shorter, because it can create a stop codon somewhere with the addition, also it can make it longer, a stop codon no longer present where it should be.\n\nBasically the protein would be non-functioning.