Progress 10/01/02 to 09/30/06
Outputs
We previously produced a line of bovine embryonic fibroblast cells (BEF) from a 40 day-old Holstein fetus and a second line from an outbred free-range Montana beef fetus. From the Holstein fetus, we prepared a genomic DNA library in lambda phage. We expanded the BEF cell line to second passage, and froze several hundred vials of these early-passage cells in liquid nitrogen to provide an essentially permanent stock of BEFs that genetically match our genomic library perfectly. The primary library had over 10-fold coverage of the genome with an average insert size of over 22kb. We isolated 5 pure clones of the bovine tbp gene form the library, which have been used for making targeting vectors. We chose this gene as a representative paradigm largely because we have great experience in working with targeting this gene in mouse cells. Using several of these vectors, we initiated targeting of the matched Holstein cells and found that the locus could be effectively targeted,
but cell migration precluded efficient clonal selection. We switched to a fluorescence-based double selection targeting method; however our studies using this failed to yield targeted clones. Analyses suggest that the fluorescence of single-copy transformed cells, as cells bearing targeted alleles should be, is too low to efficiently isolate, so only non-targeted multi-copy transfected cells are recovered by FACS. To surmount this problem, we switched to a strategy that is independent of fluorescent markers and will not exclude single-copy transformants. Cells are transfected with a targeting vector that contains only the drug selection marker (neo) and are being plated in 96-well dishes containing selective medium at densities that give 0.6 transfected cells per well. As such, most wells with cell growth are clonal derivatives of a single transfected founder, we eliminate pre-selection for high-expressers (i.e., multi-copy transformants), and all progeny of that founder are confined
to a single well, thus precluding the previous complications due to unrestricted migration. We have also vastly improved our transfection efficiency by switching from electroporation of the cells (which gave only a small percentage of the cells transfected in analyses based on green fluorescent protein expression) to using the Amaxa Nucleofector II electro-transformation system. This modification, increased our transfection efficiency by roughly 10-fold. Using this approach,w we have now isolated genomic DNA from roughly 300 clones of G418-resistant Holstein (isogeneic) BEFS and a similar numer of Angus (mismatched) BEFS. We are currently optimizing protocols for measuring homologous gene targeting vs random insertion in these clones.
Impacts
Our goal is to measure how sequence identity parameters affect the efficiency of targeted mutagenesis in bovine cells. Were targeted mutagenesis to become tractable in cattle, it could both increase the value of existing cattle-based commodities and allow creation of countless more valuable cattle-based products. Thus, it could allow production of particularly disease resistant herds or herds exhibiting increased milk or beef production. It could also help create cattle lines custom fit to particular environments, for example, by altering their ability to use different feed sources, altering salt tolerance, or altering reproductive cycles.
Publications
- Prigge JR, Schmidt EE. Interaction of protein inhibitor of activated STAT (PIAS) proteins with the TATA-binding protein, TBP. J Biol Chem. 2006 May 5;281(18):12260-9. PMID: 16522640.
- Sansinena MJ, Taylor SA, Taylor PJ, Schmidt EE, Denniston RS, Godke RA. In vitro production of llama (Lama glama) embryos by intracytoplasmic sperm injection: Effect of chemical activation treatments and culture conditions. Anim Reprod Sci. 2006 Jul 15; [Epub ahead of print] PMID: 16846701.
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Progress 01/01/05 to 12/31/05
Outputs
In founding work for this project, we produced a line of bovine embryonic fibroblasts (BEFs) from a Holstein fetus and a second line from an outbred free-range Montana beef fetus. From the Holstein fetus, we prepared a genomic DNA library in lambda phage. We expanded the line to second passage and froze vials in liquid nitrogen to provide a stock of BEFs that genetically match our genomic library perfectly. The primary library had over 10-fold coverage of the genome with an average insert size of over 22kb. We isolated 5 pure clones of the bovine tbp gene form the library, which have been used for making targeting vectors. Using these vectors, we initiated targeting of the matched Holstein cells and found that the locus could be effectively targeted, but cell migration precluded efficient clonal selection. In the past year, we improved our transfection efficiency in the past year by switching from electroporation of the cells (which gave only a small percentage of the
cells transfected in analyses based on green fluorescent protein expression) to using the Amaxa Nucleofector II electro-transformation system. This modification, has increased our transfection efficiency by roughly 10-fold. We previously had been using a fluorescence-based double selection targeting method; however our studies using this failed to yield targeted clones. Analyses suggest that the fluorescence of single-copy transformed cells, as cells bearing targeted alleles should be, is too low to efficiently isolate, so only non-targeted multi-copy transfected cells are recovered by FACS. To surmount this problem, we switched to a strategy that is independent of fluorescent markers and will not exclude single-copy transformants. Cells are transfected with a vector that contains only the drug selection marker (neo) inserted into a non-disruptive region of the bovine tbp sequence to allow homologous gene targeting, and G418-resistant clones are being plated in 96-well dishes
containing selective medium at densities that give 0.6 transfected cells per well. As such, most wells with cell growth will be clonal derivative of a single transfected founder, we will eliminate pre-selection for high-expressers (i.e., multi-copy transformants), and all progeny of that founder will be confined to a single well, thus precluding the previous complications due to unrestricted migration.
Impacts
Our goal is to measure how sequence identity parameters affect the efficiency of targeted mutagenesis in bovine cells. Were targeted mutagenesis to become tractable in cattle, it could both increase the value of existing cattle-based commodities and allow creation of countless more valuable cattle-based products. Thus, it could allow production of particularly disease resistant herds or herds exhibiting increased milk or beef production. It could also help create cattle lines custom fit to particular environments, for example, by altering their ability to use different feed sources, altering salt tolerance, or altering reproductive cycles.
Publications
- TA Tucker, JA Kundert, AA Bondareva, and EE Schmidt (2005). Reproductive and neurological quaking(viable) phenotypes in a severe combined immune deficient mouse background. Immunogenetics, 57, 226-231.
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Progress 01/01/04 to 12/31/04
Outputs
Our goal is to measure how critical it is to use isogeneic vectors for targeting mutations into bovine cells. Were targeted mutagenesis to become tractable in cattle, it could both increase the value of existing cattle-based commodities and allow creation of countless more valuable cattle-based products. Thus, it could allow production of particularly disease resistant herds or herds exhibiting increased milk or beef production. It could also help create cattle lines custom fit to particular environments, for example, by altering their ability to use different feed sources, altering salt tolerance, or altering reproductive cycles.
Impacts
Our goal is to measure how sequence identity parameters affect the efficiency of targeted mutagenesis in bovine cells. Were targeted mutagenesis to become tractable in cattle, it could both increase the value of existing cattle-based commodities and allow creation of countless more valuable cattle-based products. Thus, it could allow production of particularly disease resistant herds or herds exhibiting increased milk or beef production. It could also help create cattle lines custom fit to particular environments, for example, by altering their ability to use different feed sources, altering salt tolerance, or altering reproductive cycles.
Publications
- Tucker, T.A., J.A. Kundert, A.A. Bondareva, and E.E. Schmidt (2005). Reproductive and neurological quakingviable phenotypes in a severe combined immune deficient mouse background. Immunogenetics, in press.
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Progress 01/01/03 to 12/31/03
Outputs
Targeted mutagenesis can be used to create genetically modified animals; however, in species other than mice, there are very few reported successes (one sheep and one pig have been reported in peer reviewed papers) to date. The proposed research is aimed at increasing the efficiency of targeted mutagenesis in bovine cells, which could in turn be used to create animals by cloning. We produced bovine cell lines from a pure-bred breed of dairy cattle (American Holstein) and from an unrelated breed (free-range beef cattle from Montana). DNA from the Holstein cells has been used to produce vectors for targeted mutagenesis. Differences in targeting efficiency between the cell lines will be correlated to the number of DNA sequence differences between the two cell lines at the targeting site. We will then introduce mutations into the targeting vector and measure the effects of these on targeting efficiency in the two cell lines. As the first systematic study to analyze
parameters affecting targeted mutagenesis in bovine cells, we have produced a line of bovine embryonic fibroblast cell (BEF) from a single 40 day-old bovine fetus that we harvested from a "pure-bred" American Holstein heifer inseminated with pure American Holstein semen. The rationale for this breed choice was to minimize heterozygosity in the resultant fetus. From the same fetus, we prepared a genomic DNA library in lambda phage. We expanded the BEF cell line to second passage, and froze several hundred vials of these early-passage cells in liquid nitrogen to provide an essentially permanent stock of BEFs that genetically match our genomic library perfectly. Characterization of the library indicate the primary library had over 10-fold coverage of the genome with an average insert size of over 22kb. We have screened this library and isolated 5 pure clones of the bovine tbp gene, which will form the target for our studies. We chose this gene as a representative paradigm largely because
we have great experience in working with targeting this gene in mouse cells. Last year, we proposed that, since BEFs are highly migratory, they are poor colony formers, we would attempt to implement a rapid fluorescent marker/FACS-based method of identifying transfected clones. We have done this and it is working very well. Thus, a new vector design with both fluorescent and drug resistant markers is used for gene targeting. Several days after electroporation, fluorescent cells are isolated by FACS and plated at clonal densities in 96-well dishes. Conditions have been established to give only 0.66 transfected cells per well, so most colonies are clonal. In the next year, we will use this technology to isolate large numbers of transfected clones and use these to measure targeting efficiency.
Impacts
Our goal is to measure how critical it is to use isogeneic vectors for targeting mutations into bovine cells. Were targeted mutagenesis to become tractable in cattle, it could both increase the value of existing cattle-based commodities and allow creation of countless more valuable cattle-based products. Thus, it could allow production of particularly disease resistant herds or herds exhibiting increased milk or beef production. It could also help create cattle lines custom fit to particular environments, for example, by altering their ability to use different feed sources, altering salt tolerance, or altering reproductive cycles.
Publications
- Schmidt, E.E. AA Bondareva, JR Radke, and MR Capecchi (2003). Fundamental cellular processes do not require vertebrate-specific sequences in the TATA-binding protein. J. Biol. Chem. 278; 6168-6174.
- Bondareva, A.A., and E.E. Schmidt (2003). Early vertebrate evolution of the TATA-binding protein, TBP. Mol. Biol. Evol. 20; 1932-1939.
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