by what method may i knockout the dkk1 gene in cows?
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First you need to identify on which BAC(s) the dkk1 gene resides then order it. You then need to cut out the sections on either side of the gene or an exon - preferably about 2kb on one side and 6-8kb on the other. Once you have those pieces in your targeting vector (which contains a positive selection marker - usually TK and it's promoter), you'll need to insert a negative selection marker in the long arm (the 6-8kb side). This is usually the Neomycin resistance gene, but others have been using Puramycin among others. Once that's made, you need to open the vector, and submit it to a transgenic facility that works on cows (good luck!).\n\nThe facility will electroporate your DNA into cow stem cells and then you'll have to screen all the clones that survive the positive and negative selection (you're looking at screening at least 500 colonies and probably much more than that). Using PCR as a first pass screening method can reduce your overall workload, but you'll still have to show a shift by Southern Blot since it's still the "gold standard". Once you've idenified a positive clone, you let the transgenic facility know, and they remove the nucleus and inject it into a denucleated embryo which is then implanted into a female.
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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2835075&dopt=Abstract\n\nhttp://jpet.aspetjournals.org/cgi/content/abstract/235/3/846
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Yes, try to this!\n\nhttp://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Abstract&list_uids=3906008
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http://www.harpercollege.edu/mhealy/g101ilec/samer/smd/smphys/smdphyfr.htm\nhttp://www.dowslane.org/animal.htm\n\nIf these web sites don't help just email me and I will try to find you something different.
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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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Check out this site.\nhttp://www.universityobgyn.com/kegal.htm
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Study of "Mad Cow" disease.  Or maybe something on insulin and diabetes.
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http://www.medicinenet.com/alkaptonuria/article.htm\n\nI have not the answer,but that site is dedicated to your question
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1. Took a Cell from Dolly, I believe it was a skin cell not sure.\n2. Took an egg cell (oocyte) from a donor. Took out it nucleus.\n3. Put nucleus from the cell he got from dolly and then put it in the enucleated oocyte.\n4. Activate it, grow in culture. If successful, implant it in a female\nHe had to do this thousands of time before it worked. Look up DNA imprinting if you want to know why it doesn't work as great.
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There are many procedures with which to trypsinize cells. All include washing the cell monolayer with TD, or in rare cases, with VE. This removes serum, which contains trypsin inhibitors, and lowers the concentration of both calcium and magnesium, both of which inhibit trypsinization.\n\nI like to rinse the cells with TD, add approximately 1 ml of straight trypsin, rock the dish, aspirate a majority of the trypsin, and then incubate the dish at 37 °C until the cells come off the dish.\n\nSome people put 1 ml of 5 fold diluted, with TD, trypsin on the dish and leave it on. Others do this using full-strength trypsin.\n\nCells vary enormously in how fast they come off the dish. Some cells come off within one minute, some require 15 min. Cells should be trypsinized until they come off the dish, but not longer. Don't rush, but don't over do it.\n\nCells that are extremely hard to dislodge with trypsin should be rinsed with VE prior to trypsinization and then trypsinized in the presence of VE. The EDTA promotes disaggregation. Unfortunately, EDTA is toxic to cells at high concentration--it deprives them of Zn++.\n\nIt is essential to inactivate trypsin and to remove EDTA after you have gotten the cells off the dish. Trypsin inhibition is accomplished in part by trypsin inhibitors in the serum in the growth medium. Bovine serum is a rich source of trypsin inhibitors.\n\nThe best, but most laborious way to reseed cells, is to dilute the suspended cells with medium containing serum and spin them down in the gray table top centrifuge--5/8 speed, 4 min. Then aspirate the supe, resuspend the cells in medium, and reseed them.\n\nA short cut that works with cells that are being seeded at a low cell density is simply to dilute the trypsinized suspension of cells with medium and seed the cells directly.\n\nIf the cells are to be reseeded at a high cell density, the trypsin will not be diluted or inactivated sufficiently and the cells will not attach.\n\nThis is not satisfactory when VE is used because EDTA is growth inhibitory even at low concentrations.\n\nIt is also not satisfactory with chick cells, since they are grown in medium containing only 2% serum and cannot be seeded very sparsely.
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The success rate of invitro fertilization in cattle is very high. They have been doing it for years.
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Pink, They bleed inside the udder !
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Yes it can be done one place that does it is called questgen forensics. Also Animal DNA Services \nVeterinary Diagnostics Center does dna testing for genetic issues your pet could have and dna banking stuff like that.
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Yes I have worked with RNA before. The main difficulty with all of the procedures is making sure you have gotten rid of the RNAses. They are very tough and stable, so you have to make sure everything has RNAse inhibitors (DEPC). I dont know enought about guinea fowl to know if they use similar polyA tails that humans, etc do. If not, the oligo-dT resin wouldnt have much to bind to. I dont know what your goal is but you could consider a PCR strategy to amplify what you are looking for. If you are looking for some tissue expression, you could look into buying premade mRNA from a company, which we used successfully before. hope that helps!
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these might help..\n\nhttp://www.blackwell-synergy.com/doi/abs/10.1111/j.1365-2621.2006.01347.x\n\nhttp://www.co-lipidology.com/pt/re/colipidology/abstract.00041433-200502000-00009.htm;jsessionid=Fh8fr0yV02g1pkF1LGYLXyzy9lPXZ4Gn69QZKHJ1GG1vJYGw1czy!-1640309041!-949856145!8091!-1\n\nhttp://scholar.google.com/scholar?hl=en&lr=&q=cache:yYCPrA4fU54J:bmc.ub.uni-potsdam.de/cgi-bin/show.pl%3F1475-2891-4-33/1475-2891-4-33.pdf+list+of++biochem+experiments,+TEAC+assay+or+the+phenolics+test
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Possible, but not probable.\n\nIf one looks at the variations of canines and felines that have been developed through human breeding problems you do get the idea that this may be possible.\n\nHowever, domestic cattle have been associated with humans for a very long time and there seems to be a very much smaller difference between the variations.  In part, this is due to the goals being different (meat production, milk production, smaller horns).\n\nWhat you are talking about is a process that might take centuries and there must be a clear cut goal and reason to require such an expenditure of resources.\n\nIt sound like you want to reduce a typical weight of 1,200 to 1,800 pounds down to 80.  This may not be possible.
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Dr. Joel Wallach, B.Sc., DVM, ND\n\nCheck the sources below for more info.\n\nHope this helps.