Progress 02/19/09 to 02/18/14
Outputs
Target Audience: Other Scientific investigators and general public. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Three graduate students have participated in this project which served as partial requirement for them to obtain their PhD or MS degree. Gail Williamson, Michael Peoples and Matthew Synder. How have the results been disseminated to communities of interest? The results of our work has been published in publically accessible journals and/or presentations at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This past, and last year of this project, my laboratory group continued to focus on the development and application of genetic modification in livestock to benefit animal agriculture, animal and human health. Over the past 2 years we focused on the utilization of lentiviral vectors to produce numerous genetically modified (GM) animals including sheep, goats, pigs and cattle. Our major goal has been to produce livestock that are resistant to disease, (in particular Foot and Mouth Disease), and/or exhibit increased muscle development. Due to concerns regarding the utilization of viral vectors to produce GM livestock, and potential for unwanted effects including disease due to genetic recombination with other viruses, we also conducted studies to demonstrate their safety. However, with continuing uncertainty surrounding the safe use of viral vectors for genetic modification of livestock, we also began exploring alternative approaches for genetic modification. These efforts included the production and utilization of Talens and CRISPRs which represent new molecular tools that can be injected into one-cell stage embryos, and have been shown to cause specific mutations in targeted genes (targeted gene editing). The great majority of studies to date have been conducted in mice. Therefore, in collaboration with colleagues at the University of Minnesota, Dr. Scott Farenkrug and Dan Carlson, we used Talens to produce the world’s first GM calf which exhibits a targeted mutation in the myostatin gene. In addition to our work involving the production of live GM animals, we also devoted an enormous amount of time employing an RNA interference approach to identify and characterize key genes thought to be important for normal embryonic development. Our focus has been on those genes involved with epigenetic reprogramming during early development and epigenetic control of gene expression. Again, the majority of the studies conducted to date involve mice, and studies in other species including livestock, represent a critical need. Much of our activity this past year involved experiments in which siRNAs designed to silence the expression of specific target genes were injected into one-cell bovine embryos. The genes targeted for down regulation were selected based on their known involvement in the epigenetic regulation of gene expression, as previously determined in mouse studies. Several thousand bovine one-cell embryos were produced and injected with siRNAs designed to silence the expression of G9a, Dnmt3a, Ash2l or SetB1. Non-injected control embryos, in addition to one-cell embryos injected with a null (non-sense) siRNA were also produced. Development rates were recorded for each treatment and control group. Embryos were then fixed and prepared for analysis by ICC or further analysis by RT-PCR to determine whether or not known epigenetic marks were modified in response to down regulation of our target genes. Development rates were compared between different treatments and controls. Results are depicted in Table 1. Table 1. Development rates of bovine embryos injected with siRNAs targeting G9A, DNMT3A, ASH2L, and SETDB1 for suppression compared to grouped controlled embryos. Type of Embryo # of Embryos # Cleaved Cleave % # Blastocyst Blastocyst % Control 1647 1412 85.7% 542 32.9% NULL 2005 1587 79.2% 445 22.2% G9A 1184 991 83.7% 0 0.0%* DNMT3A 1057 895 84.7% 29 2.7%* ASH2L 466 404 86.7% 146 31.3% SETDB1 361 308 85.3% 0 0.0%* * indicates (p<.05) The suppression of G9A and SETDB1 resulted in complete developmental arrest with no viable embryos reaching the blastocyst stage. Suppression of DNMT3A resulted in minimal development (2.7% blastocysts) but still resulted in a significant loss of viability at the 8-16 cell stage of development. However, inhibition of ASH2L resulted in little variation in blastocyst development between control embryos (31.3% ± 2.0 s.e.m.,n= 466v.34.8% ± 1.9 s.e.m.,n= 418, respectively,P> 0.2), suggesting that ASH2L is less critical during the early stages of bovine embryonic development. Following siRNA injection, the embryos were cultured until the blastocyst stage or their final stage of development when they were collected for RT-qPCR or ICC . Currently, over 2000 embryos representing each of the different treatment groups have been fixed, stained, imaged and are currently waiting for ICC analysis. Samples for completing all the RT-qPCR work have also been produced and waiting for analysis. Key outcomes this past year were directly in line with our stated aims and objectives involving the utilization of genetic modification and RNA interference to study functional genomics. We have now utilized our RNAi based approach involving the injection of siRNAs into early-stage bovine embryos to assess the importance of key epigenetic regulators in supporting early embryo development. In addition we have initiated studies involving ICC and RT-PCR analysis to further characterize the mechanism of action of these (Suz12, LSH, G9A, DNMT3A, ASH2L, and SETDB1) on regulating methylation, acetylation, and expression of numerous genes previously shown critical for early embryonic development to proceed normally (SetDb1, Hp1, DNMT3a, DNMT3b, Oct4, Nanog, Sox2, CDX2, and others).
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Burroughs CA, Williamson GL, Golding MC, Long CR. Oxidative stress induced changes in epigenetic modifying gene mRNA in pre-implantation bovine embryos. 2013 Reprod Fertil Develop 25:149.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2014
Citation:
Snyder MD, Pryor JH, Peoples MD, Williamson, GL, Golding MC, Westhusin ME, Long CR. Suppression of epigenetic modifiers alters the bovine embryonic developmental program during in vitro culture. Reprod Fertil Dev. 2013 Dec; 26(1):175. PubMed PMID 24305305
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Long CR, Westhusin ME, Golding MC. Reshaping the transcriptional frontier: Epigenetics and somatic cell nuclear transfer. Mol Reprod Dev. 2013 Oct 24; PubMed PMID: 24167064
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The goal of this project is to develop genetically modified livestock which express genes encoding short-hairpin RNAs designed to silence the expression of specific genes. Our approach involves the utilization of recombinant lentiviral vectors to insert transgenes into the genome of one-cell embryos followed by embryo transfer. Using this technology both endogenous and exogenous genes can be targeted for silencing. We have produced recombinant lentiviral vectors encoding genes designed to silence the expression of DNA methyltransferase (DNMT1), Prion Protein (PrP), Myostatin (GDF 8), and Foot and Mouth Disease Virus (FMDV). Those designed to silence myostatin and FMDV were injected into one cell embryos collected from pigs, goats, sheep, and cattle. To date we have produce more than 60 genetically modified (GM) animals representing cattle, sheep, goats and pigs. using this approach. This past year we continued to focus much of our efforts on analyzing the phenotypes of these animals and initiating breeding trials to evaluate transgene inheritance and expression. Two manuscripts were published. The first involved the production of genetically modified (GM) cattle in which myostatin was targeted for silencing to increase muscle development. Although the numbers of animals involved in this study were minimal, the data indicated our approach was working, with those animals exhibiting the greatest decrease in myostatin expressing more muscle development. This work led to new collaborations with Dr. Chris Kerth and Dr. Steve Smith in the Department of Animal Sciences. As a result we have now repeated much of this work using the pig as a model and Dr. Kerth and Smith are currently analyzing muscle samples collected from this research to confirm knockdown of myostatin and increase in muscle development in pigs. We also completed and published the results of another study involving the analysis of safety issues related to the utilization of lentiviral vectors for producing transgenic sheep. This involved collaboration with Dr. Ken Cornetta from the University of Indiana and demonstrated lentiviral vectors to be a safe and effective method for producing GM livestock. In brief, numerous tissues collected from surrogate females which had received embryos injected with lentivirus were analyzed to determine if there were any signs of the transgene moving from the embryo/fetus to the surrogate. We also analyzed the potential for replication competent retrovirus to evolve due to some unanticipated recombination event with our lentivirus and endogenous sheep retroviruses. No replication competent virus was found and none of the tissues collected from surrogate mothers contained the transgene or any part of the lentiviral vector. This and other data resulting from our research was presented at various seminars, national and international meetings and published in the proceedings of the International Embryo Transfer Society meetings. Numerous graduate students presented results of this research either orally or in poster sessions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Specific outcomes and impacts of our research to date have been minimal. This is due to the current government regulations involving the development and utilization of GM Livestock for human use (production of food and medicines, etc). Thus far, only one product derived from a GM goat has been approved for human utilization and no food products have been approved by the FDA. The regulations are extremely strict. However, our demonstration that the technology we are developing is useful, and has tremendous potential to significantly impact animal agriculture, animal and human health, has resulted in our group at the Reproductive Sciences Laboratory obtaining additional funding from NIH to continue our work. Further demonstration that we can efficiently and effectively produce transgenic livestock expressing genes designed to target specific endogenous and exogenous proteins for downregulation represents a major accomplishment and has not only resulted in our ability to obtain additional funding to support our work, but increased interest from other research organizations wanting to collaborate with our group. Given the success we have experienced thus far, we anticipate the ability to continue securing the funds needed to support our work. Moreover, we believe this work will eventually result in dramatic and significant impacts on animal agriculture, animal and human health; and eventually result the production and utilization of GM livestock throughout the world.
Publications
- Tessanne K, Golding MC, Long C, Peoples M, Hannon G, Westhusin M. 2012. Production of transgenic calves expressing an effective shRNA targeting myostatin. Mol Reprod Dev.79(3): 176-85.
- Cornetta K, Tessanne K, Long C, Yao J, Satterfield C, Westhusin M. 2012. Transgenic sheep generated by lentiviral vectors: safety and integration analysis of surrogates and their offspring. Transgenic Res. (Epub ahead of print).
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The goal of this project is to develop genetically modified livestock which express genes encoding short-hairpin RNAs designed to silence the expression of specific genes. Our approach involves the utilization of recombinant lentiviral vectors to insert transgenes into the genome of one-cell embryos followed by embryo transfer. Using this technology both endogenous and exogenous genes can be targeted for silencing. We have produced recombinant lentiviral vectors encoding genes designed to silence the expression of DNA methyltransferase (DNMT1), Prion Protein (PrP), Myostatin (GDF 8), and Foot and Mouth Disease Virus (FMDV). Those designed to silence myostatin and FMDV were injected into one cell embryos collected from pigs, goats, sheep, and cattle. To date we have produce more than 60 genetically modified (GM) animals representing cattle, sheep, goats and pigs. using this approach. This past hear we focused much of our efforts on analyzing the phenotypes of these animals and initiating breeding trials to evaluate transgene inheritance and expression. One manuscript was published which involved our work with silencing the expression of DNMTs and another has been recently accepted for publication which involves the production of transgenic cattle in which myostatin was targeted for silencing to try and increase muscle development. Other data and results of this research was presented at various seminars, national and international meetings and published in the proceedings of the International Embryo Transfer Society meetings. Numerous graduate students presented results of this research either orally or in poster sessions. PARTICIPANTS: This past year we established a new collaborative effort with Dr. Chris Kerth and Dr. Steve Smith from the Department of Animal Sciences, Texas A&M University. Dr. Smith and Dr. Kerth are both interested in muscle development and are assisting us in the analysis of transgenic livestock in which myostatin has been targeted for silencing. We anticipate this new collaboration will result in the development and submission of several new grant applications to USDA and NIH. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Thus far, specific outcomes and impacts have been minimal. However, this past year we did publish several new peer-reviewed papers which represent the first significant outcomes/impact of our research efforts. Our demonstration that we can efficiently and effectively produce transgenic livestock expressing genes designed to target specific endogenous and exogenous proteins for downregulation represents a major milestone and has resulted in our ability to obtain additional funding for our research. In addition, several collaborative research projects have been initiated with other research institutions, and with commercial entities. As inticated above, we are now collaborating with Dr. Chris Kerth and Dr. Steve Smith in the Department of Animal Sciences. Dr. Smith and Kerth are helping with the analysis of muscle development in transgenic animals in which myostatin has been targeted for silencing. We anticipate this new collaboration will result in the development and submission of several new grant applications which will be submitted to NIH and USDA. Given the success of our research, especially now having produced numerous GM livestock and demonstrating the efficiency of our approach, we anticipate this work will eventually result in dramatic and significant impacts on animal agriculture, including the production and utilization of GM livestock throughout the world.
Publications
- Tessanne K, Golding MC, Long CR, Peoples MD, Hannon G, Westhusin ME. 2011 Production of transgenic calves expressing an shRNA targeting myostatin. Mol Reprod Dev. Nov 18. doi: 10.1002/mrd.22007 [Epub ahead of print]. PMID: 22139943 [PubMed - as supplied by publisher].
- Romano JE, Thompson JA, Kraemer DC, Westhusin ME, Tomaszweski MA, Forrest DW. 2011. Effects of early pregnancy diagnosis by palpation per rectum on pregnancy loss in dairy cattle. J Am Vet Med Assoc. 2011 Sep 1;239(5):668-673.
- Golding MC, Williamson GL, Stroud TK, Westhusin ME, Long CR. 2011. Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development. Mol Reprod Dev. 2011 May;78(5):306-317.
- Peoples M, Golding M, Long C, Westhusin M. 2012. Development of rlentiviral vectors for male germline-specific cre recombinase expression in livestock. Reprod Dev Fertil. 24:183.
- Tessanne K, Long C, Spencer T, Satterfield, Westhusin M. 2011. Production of transgenic sheep using recombinant lentivirus microinjection of in vivo produced embryos. Reprod Dertil Dev. 23:264.
- Peoples M, Westhusin M, Tessanne K, Long C. 2011. Production of transgenic livestock using a lentivirus expressing multiple short interfering RNAs targeting foot and mouth disease virus. Reprod Fertil Dev. 23:109.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The goal of this project is to develop genetically modified livestock which express genes encoding short-hairpin RNAs designed to silence the expression of specific genes. Our approach involves the utilization of recombinant lentiviral vectors to insert transgenes into the genome of one-cell embryos followed by embryo transfer. Using this technology both endogenous and exogenous genes can be targeted for silencing. We have produced recombinant lentiviral vectors encoding genes designed to silence the expression of DNA methyltransferase (DNMT1), Prion Protein (PrP), Myostatin (GDF 8), and Foot and Mouth Disease Virus (FMDV). Those designed to silence myostatin and FMDV were injected into one cell embryos collected from pigs, goats, sheep, and cattle. To date we have produce more than 60 genetically modified (GM) animals representing cattle, sheep, goats and pigs. using this approach. The phenotypes of these animals are currently being evaluated. This research has resulted in several invention disclosures being filed to protect intellectual property. Investigations are ongoing to determine what if any patents should be filed. Some of the data and results of this work has been presented at various seminars, national and international meetings. Abstracts have been published in the proceedings of the International Embryo Transfer Society meetings. Numerous graduate students presented results of this research either orally or in poster sessions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Thus far, specific outcomes and impacts have been minimal. However, our demonstration that we can efficiently and effectively produce transgenic livestock expressing genes designed to target specific endogenous and exogenous proteins for downregulation represents a major milestone and has resulted in our ability to obtain additional funding for our research. In addition, several collaborative research projects have been initiated with other research institutions, and with commercial entities. Given the success of our research, especially now having produced numerous GM livestock and demonstrating the efficiency of our approach, we anticipate this work will eventually result in dramatic and significant impacts on animal agriculture, including the production and utilization of GM livestock throughout the world.
Publications
- Suppression of bovine and caprine myostatin expression in vitro using RNA interference. Texas Genetics Society Annual Meeting, April 2-4, 2009.
- Long C, Tessanne K, Golding M. Applications of RNA interference-based gene silencing in animal agriculture. Reproduction, Fertility, and Development. 2009 Dec;22(1):47-58.
- K Tessanne, J Yao, K Cornetta, M Westhusin, T Spencer, C Long. 2010. Replication competent lentivirus (RCL) analysis in recipient animals of transgenic embryos produced by lentiviral transfer. International Embryo Transfer Society Annual Meeting, Cordoba, Argentina.
- K. Tessanne, C. Long, M. Golding, and M. Westhusin. 2009. Comparison of different methods for producing transgenic livestock with reduced myostatin expression using RNA interference. Texas Forum on Reproductive Sciences, Houston, Texas.
- Development of transgenic livestock for reduced myostatin expression using RNA interference. International Embryo Transfer Society Annual Meeting, January 4-6, 2009.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The goal of this project is to develop genetically modified livestock which express genes encoding short-hairpin RNAs designed to silence the expression of specific genes. Our approach involves the utilization of recombinant lentiviral vectors to insert transgenes into the genome of one-cell embryos followed by embryo transfer. Using this technology both endogenous and exogenous genes can be targeted for silencing. This past year we produced recombinant lentiviral vectors encoding genes designed to silence the expression of DNA methyltransferase (DNMT1), Prion Protein (PrP), Myostatin (GDF 8), and Foot and Mouth Disease Virus (FMDV). Those designed to silence myostatin and FMDV were injected into one cell embryos collected from pigs, goats, sheep, and cattle. Currenlty we have numerous confirmed pregnancies and the birth of genetically modified offspring is expected this spring. Results of this research have resulted in several invention disclosures and plans to file patents to protect intellectual property as soon as the offspring are born. Some of the data and results of this work has been presented at various seminars, national and international meetings. Abstracts have been published in the proceedings of the International Embryo Transfer Society meetings. Numerous graduate students presented results of this research either orally or in poster sessions. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Thus far, specific outcomes and impacts of this research have been minimal. However, given the success of our research we anticipate this work will eventually result in dramatic and significant impacts on animal agriculture, including the production and utilization of GM livestock throughout the world.
Publications
- No publications reported this period
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