Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This past year we have continued to conduct numerous experiments focused on developing improved technology for animal cloning and producing genetically engineered livestock. Recombinant lentiviral vectors containing genes encoding shRNAs that target PrP for silencing were produced and utilized to infect bovine fibroblasts. The resulting transgenic bovine cell lines have been screened using in vitro assays and significant knockdown (>80%) of genes coding for PrP was demonstrated. These cell lines will be utilized for nuclear transfer to produce cloned transgenic embryos. We then plan to transfer these embryos into synchronized recipient cows for the production of transgenic fetuses which will be compared to control fetuses to confirm significant knockdown of PrP. Additional progress has been made towards targeting genes encoding myostatin for silencing by RNA interference. Silencing this gene should result in increased muscle and meat development in transgenic animals.
Recombinant lentiviral vectors were produced and utilized to infect HEK 293 cells with an shRNA designed to silence myostatin. These cells were then transfected with a CMV plasmid expressing myostatin linked to GFP. Subsequent analysis of the transgenic cells indicated approximately 70% knockdown of myostatin gene expression. The same recombinant lentivirus has now been utilized to produce transgenic goat fibroblast cells that are being utilized for nuclear transfer to produce cloned transgenic goats. Numerous cloned transgenic embryos have been transferred into recipient does, however to date we have not obtained any pregnancies. Significant progress was also made involving efforts focused on silencing the expression of DNMT1 by RNA interference in cell lines and embryos. Recombinant lentiviruses encoding shRNAs targeting DNMT1 for silencing were utilized to infect bovine fetal fibroblasts. Analysis of these cells indicated a significant knockdown of DNMT1 gene expression. These
cells will now be utilized for nuclear transfer to produce cloned offspring. The goal of this research is to characterize the role of DNMT1 during early development. In addition, we anticipate silencing of this gene in cell lines utilized for nuclear transfer may result in an increase in the efficiency of cloning. Results of this research have been presented at numerous conferences and scientific meetings including the International Embryo Transfer Society Meetings and the Society for the Study of Reproduction Meetings. At least 4 different graduate students and 2 postdoctoral trainees have participated in this work. New research collaborations have been established with Dr. Peter Mason at the University of Texas Galveston, Viagen Inc., Austin Texas, and Transova Genetics, Souix Center, Iowa.
PARTICIPANTS: Mark Westhusin served as the Principal investigator on this research project. His responsibilities included overall management of the research activities in addition to mentoring graduate students and postdoctoral fellows. Charles Long served as a co-investigator on this research project. Dr. Long helped with the design and production of shRNAs in addition to other molecular biology work as necessary. Dr. Long also participated in mentoring graduate students. Gayle Linger is an undergraduate genetics student who has recently joined the laboratory and will be pursuing a graduate degree. Gayle has been involved in numerous aspects of this research project including the production and screening of shRNAs in addition to gene expression analysis. Carol Hanna is a PhD student in our laboratory. The majority of her work has involved investigations into different methods for delivery of siRNAs and shRNAs into both cell lines and early stage embryos. Kim Tessane is a PhD
graduate student working in the lab. Kim's focus involved work pertaining to the the design, production and screening of shRNAs. She was also responsible for maintaining the goat herd in addition to planning and scheduling all animal work. Mike Peoples is a PhD graduate student working in the lab. Mike's focus has involved all work pertaining to the production of transgenic livestock in which PrP has been targeted for silencing Sarah Canterberry is a postdoctoral fellow working in the lab. Sarah assisted numerous graduate and undergraduate students in various aspects of their projects.
TARGET AUDIENCES: Oral Presentations: Cloning of food animals and endangered species. Presented by Dr. Mark Westhusin at the Society for the Study of Reproduction Annual Meeting July 21 -25, 2007, San Antonio. Transgenic approaches to disease resistant livestock. Presented by Dr. Charles Long at the 2nd Symposium for Agriculture Biotechnology Risk Analysis, December 5-6, 2007, Research College Park, MD Animal cloning and transgenics: Applications of RNA interference. Presented by Dr. Charles Long at the University of Texas San Antonio Molecular and Cellular Biology Forum, Host: Dr. John McCarrey, October, 2007, San Antonio, Tx Disease resistance in transgenic livestock using RNA interference. Presented by Dr. Charles Long at the Transgenic Animal Research Conference VI , August, 2007, Lake Tahoe, CA, RNA interference: A new method to study functional genomics in livestock. Presented by Dr. Mark Westhusin at the 2007 Intermountain Systems Biology Symposium, June 5-7, 2007, Utah State
University, Logan Utah. Poster Presentations: Lentiviral mediated transgenic fetal bovine fibroblast cells expressing shRNAs targeting the prion protein. Michael Peoples, Charles Long, Mark Westhusin. Advances in Transgenic Animal Research, January 11-13, 2008 Houston Texas. Transfection of bovine oocytes and pre-implantation embryos with commercial transfection reagents: comparison of embryonic stage and zona integrity on transfection efficiency. Carol Hanna, Charles Long, Mark Westhusin, Duane Kraemer. Society for the Study of Reproduction annual meeting July 21 -25, 2007, San Antonio Texas Development of transgenic livestock with reduced myostatin expression using RNA interference. Kim Tessanne, Charles Long, Mark Westhusin. Texas Forum for Reproductive Sciences, April 20 - 21 2007, Houston Texas
Impacts
The initial goal of this research project was to conduct research focused on improving the efficiency of producing cloned livestock by nuclear transfer. Over time, the goals of the research expanded to include improving methods for producing transgenic livestock. To this end we have established repeatable methods for producing recombinant lentiviruses that can be utilized to effectively produce transgenic cell lines and embryos. We are currently transferring these embryos into recipient cattle or goats to try and produce transgenic embryos. In addition we have developed successful technologies involving the utilization of RNA interference to silence the expression of specific genes. Genes that have been selected for targeting include myostatin, PrP, and DNMT1. Reduction in myostatin should result in animals exhibiting increased muscle development. Reduction in PrP should result in animals resistant to TSE type diseases. Reduction in DNMT1 should result in an overall
improvement in embryo/fetal development of animals produced by nuclear transfer. New collaborations have been established with two commercial companies, Transova Genetics Souix Center Iowa and Viagen Inc., Austin Texas. Through the course of this research, numerous changes were made regarding plasmid and gene construct design to test different promoters and markers of gene expression. New protocols were also developed for the production and concentration of recombinant lentiviruses. This has resulted in significant improvements in the efficiency of the techniques we employ. We have continued to make significant progress towards the development of technology for producing transgenic animals resistant to disease and or other improved production characteristics and anticipate our research findings will have a significant impact on the future of agriculture and the important role genetically engineered livestock will play.
Publications
- Hanna, C., Long, C., Westhusin, M., and Kraemer, D. 2007. Transfection of bovine oocytes and pre-implantation embryos with commercial transfection reagents: comparison of embryonic stage and zona integrity on transfection efficiency. Proceedings of Society for the Study of Reproduction Annual Meeting July 21 -25, San Antonio, Texas (P 112).
- Westhusin, M., Kraemer, D., and Long, C. 2007. Cloning of food animals and endangered species. Proceedings of Society for the Study of Reproduion Annual Meeting July 21 -25, San Antonio, Texas (P 61).
|
Progress 05/04/01 to 03/03/07
Outputs
OUTPUTS: Throughout the course of this project we conducted numerous experiments focused on developing improved technology for animal cloning and producing genetically engineered livestock. Significant progress was made involving efforts focused on trying to determine the role of methylation during early bovine development by silencing the expression of DNMT1 using RNA interference in cell lines and embryos. Recombinant lentiviruses encoding shRNAs targeting DNMT1 for silencing were utilized to infect bovine fetal fibroblasts. Analysis of these cells indicated a significant knockdown of DNMT1 gene expression. These cells were then utilized for nuclear transfer to try and produce cloned offspring. Some early pregnancies were obtained but all of these were subsequently lost. These experiments indicated that when using a transgenic cell line in which DNMT1 was down-regulated, for nuclear transfer, blastocysts could be produced but establishment of pregnancy failed. In other studies to try and determine the role of DNMT1 on early development, we produced early stage (1-cell) bovine embryos and injected these with an siRNA designed to silence the expression of DNMT1. These embryos showed a marked decrease in development to the blastocyst when compared to control embryos, indicating that DNMT 1 was required for normal bovine early embryo development to proceed. Recombinant lentiviral vectors containing genes encoding shRNAs that target the prion protein (PrP), myostatin and Foot and Mouth Disease Virus (FMDV) for silencing were produced and utilized to infect bovine fibroblasts. The The resulting transgenic bovine cell lines were screened using in vitro assays and significant knockdown in expression of the targeted genes was demonstrated. These cell lines will be utilized for nuclear transfer to produce cloned transgenic embryos. Several pregnancies were produced however these were subsequently lost around 60 days of gestation. Three non-transgenic control pregnancies were produced and the fetuses collected for analysis. These will be utilized for controls in future experiments. The goal of this research is to characterize the role of DNMT1 during early development. We anticipate silencing of this gene in cell lines utilized for nuclear transfer may result in an increase in the efficiency of cloning. Initial studies involving both cloning and injection of early stage embryos indicate DNMT1 plays a critical role in early bovine early development. Data collected thus far still indicate we may see an improved development rate in cloned embryos if we treat the cell lines used for cloning with RNAi approaches to silence the expression of DNMT1. However, it is important to point out that this knockdown in expression needs to be transient in nature and needs to occur in the cells that are being used for cloning. 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
The initial goal of this research project was to conduct research focused on improving the efficiency of producing cloned livestock by nuclear transfer in addition to improving methods for genetically engineering livestock in which specific genes were targeted for silencing by RNAi. Over time, the goals of the research expanded to include improving methods for producing transgenic livestock. To this end we have established repeatable methods for producing recombinant lentiviruses that can be utilized to effectively produce transgenic cell lines and embryos. We are currently transferring these embryos into recipient cattle or goats to try and produce transgenic embryos. In addition we have developed successful technologies involving the utilization of RNA interference to silence the expression of specific genes. Genes that have been selected for targeting include myostatin, PrP, and DNMT1. Reduction in myostatin should result in animals exhibiting increased muscle development. Reduction in PrP should result in animals resistant to TSE type diseases. Reduction in DNMT1 should result in an overall improvement in embryo/fetal development of animals produced by nuclear transfer.
Publications
- No publications reported this period
|
Progress 01/01/06 to 12/31/06
Outputs
This past year we have continued to focus on improving methods for producing transgenic livestock in which specific genes have been targeted for silencing by RNA interference. Two separate approaches have been explored. In the first approach, a recombinant lentivirus was utilized to produce transgenic goat fibroblasts encoding green fluorescent protein (GFP) along with a short-hairpin RNA (shRNA) targeting the expression of Prion Protein (PrP). These were then utilized for nuclear transplantation to produce cloned, transgenic goat embryos, some of which were subsequently transferred into recipient does. A single pregnancy was obtained. The fetus was removed at 81 days of gestation to confirm it was transgenic and determine if PrP was successfully down regulated. Genetic analysis confirmed that the cloned fetus was expressing both GFP in addition to the shRNA targeting PrP. More relevant, when compared to a normal, age-matched goat fetus, PrP protein levels were
decreased by more than 90%. In a second approach, we attempted to utilize our recombinant lentivirus for direct injection into early stage bovine embryos. The purpose of these experiments was to determine if we could bypass the cloning process so to improve the efficiency of the entire process. Micromanipulation was utilized to inject 139 in vitro matured bovine with the recombinant lentivirus. Of these 42 (30%) developed to the blastocyst stage and of these 32 (76%) were expressing GFP, indicative that they were also expressing the shRNA targeting PrP for silencing. Results of this work were recently published in The Proceedings of the National Academy of Sciences.
Impacts
Successful and repeatable techniques for cloning and genetic engineering in livestock will have a tremendous impact on animal agriculture, animal and human health. Research completed this last year represents the first demonstration that RNA interference based technologies can be applied to livestock species to silence the expression of specific genes. Results of this work will lead to the utilization of this technology to silence the expression of many other genes and as such provide a powerful tool to study functional genomics in livestock. In the future, this technology will likely be applied to produce genetically engineered livestock with improved production traits and resistance to disease.
Publications
- Westhusin ME, Shin T, Templeton JW, Burghardt RC, Adams LG. 2006. Rescuing valuable genomes by animal cloning: a case for natural disease resistance. J. of Anim Sci. In press.
- Williams JB, Shin T, Liu L, Flores-Foxworth G, Romano J, Blue-McClendon A. 2006. Cloning of exotic/endangered species: desert bighorn sheep. Methods Mol. Biol. 348:169-182.
- Willingham-Rocky LA, Golding MC, Wright JM, Kraemer DC, Westhusin ME, Burghardt RC. 2006. Cloning of GJA1 (Connexin43) and its expression in canine ovarian follicles throughout the estrous cycle. Gene Expr. Patterns 7:66-71.
- Golding MC, Long CR, Carmell MA, Hannon GJ, Westhusin ME. 2006. Suppression of prion protein in livestock by RNA interference. Proc Natl Acad Sci 103:5285-5290.
|
Progress 01/01/05 to 12/31/05
Outputs
This past year we have continued to focus on improving methods for producing transgenic livestock in which specific genes have been targeted for silencing by RNA interference. Two separate approaches have been explored. In the first approach, a recombinant lentivirus was utilized to produce transgenic goat fibroblasts encoding green fluorescent protein (GFP) along with a short-hairpin RNA (shRNA) targeting the expression of Prion Protein (PrP). These were then utilized for nuclear transplantation to produce cloned, transgenic goat embryos, some of which were subsequently transferred into recipient does. A single pregnancy was obtained. The fetus was removed at 81 days of gestation to confirm it was transgenic and determine if PrP was successfully down regulated. Genetic analysis confirmed that the cloned fetus was expressing both GFP in addition to the shRNA targeting PrP. More relevant, when compared to a normal, age-matched goat fetus, PrP protein levels were
decreased by more than 90%. In a second approach, we attempted to utilize our recombinant lentivirus for direct injection into early stage bovine embryos. The purpose of these experiments was to determine if we could bypass the cloning process so to improve the efficiency of the entire process. Micromanipulation was utilized to inject 139 in vitro matured bovine with the recombinant lentivirus. Of these 42 (30%) developed to the blastocyst stage and of these 32 (76%) were expressing GFP, indicative that they were also expressing the shRNA targeting PrP for silencing. Results of this work were recently published in The Proceedings of the National Academy of Sciences.
Impacts
Successful and repeatable techniques for cloning and genetic engineering in livestock will have a tremendous impact on animal agriculture, animal and human health. Research completed this last year represents the first demonstration that RNA interference based technologies can be applied to livestock species to silence the expression of specific genes. Results of this work will lead to the utilization of this technology to silence the expression of many other genes and as such provide a powerful tool to study functional genomics in livestock. In the future, this technology will likely be applied to produce genetically engineered livestock with improved production traits and resistance to disease.
Publications
- Early Pregnancy Diagnosis by Transrectal Ultrasonography in Dairy Cattle. 2006. Romano, J.E., Thompson, J.A., Kraemer, D.C., Forrest, D.W., Westhusin, M.E. and M.A. Tomaszweski. Thereiogenology, In Press.
- Suppression of Prion Protein in Livestock by RNA Interference, 2006, Golding M.C., Long, C.R., Carmell, M.A., Hannon, G.J. and M.E. Westhusin. Proc. Nat. Acad. Sci., In Press.
|
Progress 01/01/04 to 12/31/04
Outputs
This past year the majority of our efforts have focused on improving methods for producing transgenic livestock. We have continued to explore the utility of lentiviral vectors for delivery of transgenes to early-stage embryos. A second approach has involved the utilization of lentiviral vectors to deliver transgenes to cells growing in culture, then select and utilize these cells for nuclear transplantation to produce cloned, transgenic livestock. The advantage of utilizing early-stage embryos as targets for the lentiviral vector is that this approach will allow for the production of transgenic livestock without the need for cloning. Cloning remains a very inefficient process, and as such, large numbers of embryos must be transferred into surrogate mothers to produce a single offspring. Unfortunately, variability in the effectiveness of our lentiviral vector delivery system has slowed progress focused on using early-stage embryos. We have however successfully
transformed goat cell lines and are currently utilizing these for nuclear transfer to produce cloned transgenic goats. Thus far we have transferred several hundered cloned embryos into recipient females. Ultrasonograpy will be employed in the coming weeks to determine if any of these are pregnant. We feel confident that we are transferring cloned embryos that are transgenic, as a gene encoding green fluorescent protein (GFP) has been incorporated into our construct, and in cases where the cloned embryos were placed into in vitro embro culture rather than being transferred into surrogate mothers, we have utilized fluorescence microscopy to confirm they are indeed expressing GFP. Genes we have selected for delivery into the goat genome encode short hairpin RNAs (shRNA). The objective here is to exploit the phenomenon of RNA interference to target and knock down the expression of either endogenous or exogenous genes. Gene constructs encode for both the GFP gene and the shRNA. Expression
of GFP is an indicator that the shRNA is also being expressed. Thus far we have produced shRNAs targetting several genes of interest, however most of our focus has involved prion protein (PrP). Previous work in mice has demonstrated that inactivation of this gene results in animals resistant to prion diseases. In goats, this disease is scrapie. As a result, a goat expressing shRNAs that knockdown the expression of PrP should be resistant to this disease. Successful demonstration of this in goats will lay the ground work for other research involving cattle with the goal of producing cattle resistant to Bovine Spongiform Encephalopathy (BSE, mad cow disease). Other endogenous genes we are currently targeting include myostatin and DNA methyltransferase. In addition we are targeting exogenous genes which encode for Foot and Mouth Disease Virus (FMDV), with the goal here being the production of livestock resistant to FMD. shRNAs targeting these genes have already been designed. They are
currently being screened for effectiveness and incorporated into lentiviral vector delivery systems.
Impacts
Successful and repeatable techniques for cloning and genetic engineering in livestock will have a tremendous impact on animal agriculture, animal and human health. Techniques can be used to preserve and then recover the genotype of valuable animals that have been deceased. Cloning can also be utilized to replicate animals with superior genotypes for use in the production of meat, milk, and fiber, and greatly improve the efficiency of genetic engineering in livestock and other animals. Scientific information generated as a result of this research project will be useful for improving the current techniques employed for cloning and genetic engineering in livestock.
Publications
- Williams J, Shin T, Liu L, Flores-Foxworth G, Romano J, Blue-McClendon A, Kraemer D,Westhusin M. 2005. Cloning of Exotic/Endangered species (Desert Bighorn Sheep). Book chapter in, Cell Reprogramming and Transgenesis by Nuclear Transfer in Vertebrates, as part of the ongoing series,Methods in Molecular Biology, published by Humana Press, USA. (in press).
|
Progress 01/01/03 to 12/31/03
Outputs
The objective of this research program is to develop and improve methods for animal cloning by nuclear transfer in addition to improving methods for genetic engineering of livestock. Different species in which cloning experiments have been conducted include dogs, cats, goats, cattle, bighorn sheep and white-tailed deer. Cloned embryos representing each of these species have been produced. The techniques utilized and the efficiency of the cloning process varies between species. We have successfully produced cloned cattle, goats, the world's first cloned cat and first white-tailed deer. We have obtained pregnancies following the transfer of cloned embryos in dogs, bighorn sheep. but have yet to produce live offspring in these species. In addition to efforts involving animal cloning we have continued to conduct experiments focused on analysis of gene expression and protein synthesis to determine whether aberrant gene expression in cloned embryos is the major cause for
high embryonic/fetal mortality. Our research has focused on characterization of genes encoding demethyltransferases (DNMTs), the key enzymes responsible for establishing genomic methylation following fertilization. Preliminary data suggests these enzymes are not functioning normally in embryos produced by somatic cell cloning and may be responsible for abnormal gene expression. Other experiments have clearly demonstrated that expression of DNMT genes is different between species. Our data indicate that gene expression profiles of DNMTs during early mammalian development are different between mice and cattle. Significant progress was also made this past year towards employing RNAi techniques to disrupt expression of the Dnmts during early bovine development. Small hairpin RNAs (shRNAs) targeted against the bovine Dnmts were designed, produced and screened for efficacy. Initial trials to test RNAi in bovine embryos were initiated. An alternative method for delivery of shRNAs into early
stage embryos, which involves the use of a lentiviral vector, was tested. Preliminary experiments indicated this system will be effective for specific gene targeting in bovine embryos. Preliminary trials using his system were conducted to deliver gene constructs expressing GFP into 1-cell bovine zygotes along with an shRNA targeting the GFP mRNA. Embryos injected with vector carrying GFP alone exhibited the expected green fluorescence while those injected with vectors carrying both the GFP and shRNAs targeting GFP displayed drastically reduced florescence. Lentiviral vector systems may allow the application of specific gene targeting at different developmental stages, thus expanding the ability to target genes during very specific times of development. It is our goal to produce recombinant lentiviral vectors containing a variety of different shRNAs targeting specific genes, then use these to determine if they are effective at 'knocking down' the expression of the targeted genes in the
resulting transgenic animals. If this proves successful, it may offer a much more effective and efficient approach for specific gene inactivation in livestock species vs standard methods involving 'knock-out' technology.
Impacts
Successful and repeatable techniques for cloning and genetic engineering in livestock will have a tremendous impact on animal agriculture, animal and human health. Techniques can be used to preserve and then recover the genotype of valuable animals that have been deceased. Cloning can also be utilized to replicate animals with superior genotypes for use in the production of meat, milk, and fiber, and greatly improve the efficiency of genetic engineering in livestock and other animals. Scientific information generated as a result of this research project will be useful for improving the current techniques employed for cloning and genetic engineering in livestock.
Publications
- Westhusin M., K. Hinrichs, Y.H. Choi, T. Shin, L. Liu and D. Kraemer. 2004. Cloning Companion Animals (Horse, Cat, Dog). Cloning and Stem cells 5:301-317.
- Golding, M.C. and M.E. Westhusin. 2003. Analysis of DNA (cytosine-5) methyltransferase mRNA sequence and expression in preimplantation embryos, fetal and adult tissues. Gene Expression Patterns 3: 551-558.
- Willingham-Rocky L.A., K. Hinrichs, M.E. Westhusin and D.C. Kraemer. 2004. Effects of stage of cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro. Reproduction (in press).
- Choi Y.H., L.B. Love, M.E. Westhusin and K. Hinrichs. 2004. Activation of equine nuclear transfer oocytes: methods and timing of treatment in relation to nuclear remodeling. Biol. Reprod 70:46-53
- Choi Y.H., Y.G. Chung, S. Walker, M.E. Westhusin and K Hinrichs. 2003. In vitro development of equine nuclear transfer embryos: Effects of oocyte maturation and amino acid composition during embryo culture. Zygote 11:77-86.
|
Progress 01/01/02 to 12/31/02
Outputs
The objective of this research program is to develop and improve methods for producing animals using assisted reproductive technologies. The primary focus involves animal cloning by nuclear transplantation. Different species in which cloning experiments have been conducted include dogs, cats, and cattle. Cloned embryos representing each of these species have been produced, however the techniques utilized and the efficiency of the cloning process varies considerably between species. Thus far we have successfully produced cloned cattle, goats and the world's first cloned cat. We have obtained pregnancies following the transfer of cloned embryos in dogs, bighorn sheep, and white-tailed deer, but have yet to produce live offspring in these species. In the past 6 months we have refocused our efforts so to concentrate on work involving cattle, goats, and white-tailed deer and are no longer working with cats and dogs.
Impacts
Successful and repeatable techniques for cloning animals will have a tremendous impact on animal agriculture, animal and human health. Techniques can be used to preserve and then recover the genotype of valuable animals that have been deceased. Cloning can also be utilized to replicate animals with superior genotypes for use in the production of meat and milk, and greatly improve the efficiency of genetic engineering in livestock and other animals.
Publications
- Long CR, Walker SC, Tang RT, Westhusin ME. 2003. New commercial opportunities for advanced reproductive technologies in horses, wildlife, and companion animals. Therio. In press.
- Choi YH, Chung YG, Walker SC, Westhusin ME, Hinrichs K. 2003. In vitro development of equine nuclear transfer embryos: effects of oocyte maturation media and amino acid composition during embryo culture. Zygote. In press.
- Choi YH, Love CC, Chung YG, Varner DD, Westhusin ME, Burghardt RC and Hinrichs K. 2002. Production of nuclear transfer horse embryos by Piezo-driven injection of somatic cell nuclei, and activation with stallion sperm cytosolic extract. Biol Reprod. 67:561-567.
- Choi YH, Shin T, Love CC, Johnson C, Varner DD, Westhusin ME, Hinrichs K. 2002. Effect of co-culture with theca interna on nuclear maturation of horse oocytes with low meiotic competence, and subsequent fusion and activation rates after nuclear transfer. Therio 57: 1005-1011.
- Otoi T, Willingham L, Shin T, Kraemer DC, and Westhusin M. 2002. Effects of oocyte culture density on meiotic competence of canine oocytes. Reproduction 124: 775-781.
- Shin T, Kraemer D, Pryor J, Liu L, Rugila J, Howe L, Buck S, Murphy K, Lyons L, Westhusin M. 2002. A cat cloned by nuclear transplantation. Nature 415:859.
|
Progress 01/01/01 to 12/31/01
Outputs
The objective of this research program is to develop and improve methods for producing animals using assisted reproductive technologies. The primary focus involves animal cloning by nuclear transplantation. Different species in which cloning experiments are being conducted include dogs, cats, and cattle. Cloned embryos representing each of these species have been produced, however the techniques utilized and the efficiency of the cloning process varies considerably between species. Dogs represent the most difficult challenge due to the inability to collect large numbers of unfertilized ova to use for nuclear transfer, and the inability to induce estrus in potential surrogate females. In the past 6 months, 19 cloned dog embryos have been transferred into 5 recipient females but no pregnancies have resulted. In contrast to our work in dogs, cloned cat embryos have been relatively easy to produce due to the ability to obtain large numbers of ova by in vitro oocyte
maturation, and induce estrus in females by hormonal manupulation. Since July of 2000, 543 cloned cat embryos have been produced and transferred into 17 different recipients. Unfortunately, even with this, we have yet to obtain a pregnancy. In cattle our work has been more encouraging. We can now routinely produce large numbers of cloned cattle embryos with the percentage of nuclear transfer embryos developing to the blastocyst stage in vitro approximating 40%. The percentage of cloned embryos transferred that results in a viable offspring is still very low. In our most recent trial, 39 blastocysts were transferred into 20 recipients resulting in 10 pregnancies at 35 days of gestation. Only one of these developed to term resulting in a healthy bull calf. The most significant aspect of this study is the cells used to produce this cloned calf were derived from a deceased black angus bull (Bull 86) that had been previously been shown to be naturally (genetically) resistant to
brucellosis. In addition, the cells had been collected and cryopreserved for 15 years prior to thawing and utilization for nuclear transfer. In cattle, pregnancy loss between day 53 and day 90 of gestation continues to me a major problem. We are continuing to conduct experiments focused on analysis of gene expression to determine whether aberrant gene expression in cloned embryos and fetuses is the major cause for high embryonic/fetal mortality. Preliminary data indicates that genes encoding insulin-like growth factors (IGFs) and their receptors are abnormally expressed in cloned embryos and fetuses when compared to normal. Genes encoding several metabolic enzymes including citrate synthetase, phosphofructo kinase, and lactate dehydrogenase have also been analyzed and appear to be normally expressed in embryos produced by nuclear transfer.
Impacts
Successful and repeatable techniques for cloning animals will have a tremendous impact on animal agriculture, animal and human health. Techniques can be used to preserve and then recover the genotype of valuable animals that have been deceased. Cloning can also be utilized to replicate animals with superior genotypes for use in the production of meat and milk, and greatly improve the efficiency of genetic engineering in livestock and other animals.
Publications
- No publications reported this period
|
Progress 01/01/00 to 12/31/00
Outputs
The objective of this research program is to develop and improve methods for producing animals using assisted reproductive technologies. The primary focus involves animal cloning by nuclear transplantation. Different species in which cloning experiments are being conducted include dogs, cats, and cattle. Cloned embryos representing each of these species have been produced, however the techniques utilized and the efficiency of the cloning process varies considerably between species. Dogs represent the most difficult challenge due to the inability to collect large numbers of unfertilized ova to use for nuclear transfer, and the inability to induce estrus in potential surrogate females. In the past 6 months, 19 cloned dog embryos have been transferred into 5 recipient females but no pregnancies have resulted. In contrast to our work in dogs, cloned cat embryos have been relatively easy to produce due to the ability to obtain large numbers of ova by in vitro oocyte
maturation, and induce estrus in females by hormonal manupulation. Since July of 2000, 543 cloned cat embryos have been produced and transferred into 17 different recipients. Unfortunately, even with this, we have yet to obtain a pregnancy. In cattle our work has been more encouraging. We can now routinely produce large numbers of cloned cattle embryos with the percentage of nuclear transfer embryos developing to the blastocyst stage in vitro approximating 40%. The percentage of cloned embryos transferred that results in a viable offspring is still very low. In our most recent trial, 39 blastocysts were transferred into 20 recipients resulting in 10 pregnancies at 35 days of gestation. Only one of these developed to term resulting in a healthy bull calf. The most significant aspect of this study is the cells used to produce this cloned calf were derived from a deceased black angus bull (Bull 86) that had been previously been shown to be naturally (genetically) resistant to
brucellosis. In addition, the cells had been collected and cryopreserved for 15 years prior to thawing and utilization for nuclear transfer. In cattle, pregnancy loss between day 53 and day 90 of gestation continues to me a major problem. We are continuing to conduct experiments focused on analysis of gene expression to determine whether aberrant gene expression in cloned embryos and fetuses is the major cause for high embryonic/fetal mortality. Preliminary data indicates that genes encoding insulin-like growth factors (IGFs) and their receptors are abnormally expressed in cloned embryos and fetuses when compared to normal. Genes encoding several metabolic enzymes including citrate synthetase, phosphofructo kinase, and lactate dehydrogenase have also been analyzed and appear to be normally expressed in embryos produced by nuclear transfer.
Impacts
Successful and repeatable techniques for cloning animals will have a tremendous impact on animal agriculture, animal and human health. Techniques can be used to preserve and then recover the genotype of valuable animals that have been deceased. Cloning can also be utilized to replicate animals with superior genotypes for use in the production of meat and milk, and greatly improve the efficiency of genetic engineering in livestock and other animals.
Publications
- Elhassan YM, Leanez AC, Tasca RJ, Wu G, Watson AJ, Westhusin ME. 2001. Amino acid levels in bovine reproductive tract and culture medium supplemented with commercial amino acid stocks. Therio. In press.
- Watson PH, Westhusin ME, Watson AJ. 2001. Expression of PTHrP and PTH/PTHrP-R mRNAs and polypeptides in bovine ovary and stimulation of bovine blastocyst development in vitro following PTHrP treatment during oocyte maturation. Anatomy and Embryology. In Press.
- Calder MD, Caveney AN, Westhusin ME & Watson AJ. 2001. Cyclooxygenase-2 and prostaglandin E2 receptor mRNAs are affected by bovine oocyte maturation time and cumulus-oocyte complex quality and prostaglandin E2 induces moderate expansion of the bovine cumulus in vitro. Biol Reprod In Press.
- Betts DH, Bordignon V, Hill JR, Winger Q, Westhusin ME, Smith LC, King WA. 2001. Reprogramming of telomerase activity and rebuilding of telomere length in cloned cattle. Proc Natl Acad Sci. In Press.
- Natale D, De Sousa PA, Westhusin ME, Watson AJ. 2001. Sensitivity of bovine blastocyst gene expression patterns to culture environments assessed by differential display-RT-PCR. Proceedings of British Society of Animal Science. In Press.
- Westhusin ME, Burghardt RC, Rugila JN, Willingham LA, Liu L, Shin T, Howe LM, Kraemer DC. 2001. Potential for cloning dogs. J Reprod. Fert. In Press.
- Westhusin ME, Long CR, Shin T, Liu L, Piedrahita JA. Cloning to reproduce desired genotypes. 2001. Therio. 55: 35-49.
- Westhusin M, and Piedrahita JA. 2000. Three little pigs worth the huff and the puff?. Nature Biotechnol. 18:1144-1145.
- Hill JR, Burghardt RC, Long CR, Looney CR, Spencer TE, Thompson JA, Winger QA, Westhusin ME. 2000. Evidence for placental abnormality as the major cause of mortality in first trimester somatic cell cloned bovine fetuses. Biol Reprod. 63:1787-1794.
- Hill JR, Winger GQ, Long CR, Looney CR, Thompson JA, Westhusin ME. 2000. Development rates of bovine nuclear transfer embryos derived from adult and fetal cells. Biol Reprod 62:1135-1140.
- Hill JR, Winger QA, Jones K, King WA, Westhusin ME. 2000. The effect of donor cell serum starvation and oocyte activation compounds on the development of somatic cell cloned embryos. Cloning 1:201-208.
- Winger QA, Hill JR, Shin T, Watson AJ, Kraemer DC, Westhusin ME. 2000. Genetic reprogramming of lactate dehydrogenase, citrate synthase, and phosphofructokinase mRNA in bovine nuclear transfer embryos produced using bovine fetal fibroblast cell nuclei. Mol Reprod Dev 56:458-464.
- Natale DR, Kidder GM, Westhusin ME, Watson AJ. 2000. Assessment by differential display-RT-PCR of mRNA transcript transitions and characterization of a-amanitin sensitive gene expression during bovine pre-attachment development by DD-RTPCR. Mol Reprod Dev. 55:152-163.
- Watson AJ, DeSousa PA, Caveney A, Barcroft LC, Natale DR, Urquhart J, Westhusin ME. 2000. Impact of bovine oocyte maturation media on oocyte transcript levels, blastocyst development, cell number and apoptosis. Biol Reprod. 62:355-364.
- Shin T, Elhassan Y, Caveney A, Watson A, Looney C, Kraemer D, and Westhusin M. 2001. Comparison of embryo survival rates and apoptotic levels in cloned bovine embryos cultured in semi-defined medium (G1G2) and chemically defined medium. Therio 55:291. Abstract.
- Winger QA, Hill JR, Shin TY, Watson AJ, and Westhusin ME. 2001. Reprogramming of IGF-II mRNA in bovine somatic cell nuclear transfer embryos and fetal liver. Therio 55:296. Abstract.
- Williams B, Keller DL, Flores-Foxworth G, Westhusin M, and Kraemer DC. 2001. In vitro fertilization of canine ova with epididymal spermatozoa. Therio 55:442. Abstract.
- Keller DL, Cox AM, Westhusin ME and Kraemer DC. 2001. Feline oocyte activation: effects on levels of MPF and MAPK. Therio 55:454 Abstract.
- Calder MD, Caveney AN, Westhusin ME, Watson AJ. 2000. The role of the Cyclooxygenase (COX-2), prostaglandin E2 (PGE2) and prostaglandin receptors 2 and 3 (EP2 and EP3) during maturation in vitro of bovine cumulus oocyte complexes (COCS). 62:271-272. Abstract.
- Elhassan YM, Zhang X, Kraemer DC, Westhusin ME. 2000. Blastocyst development of in vitro produced morula-stage bovine embryos briefly exposed to ultraviolet light following Hoechst staining. Biol. Reprod. 62: 253. Abstract.
- Liu L, Shin T, Kraemer DC, Westhusin ME. 2000. High blastocyst development following nuclear transfer with fibroblast cells derived from a cloned fetus. Biol. Reprod. 62:191. Abstract.
- Watson PH, Westhusin ME, Watson AJ. 2000. Characterization of PTHrP and PTHr in the bovine ovary. Biol. Reprod. 62:168-169. Abstract.
- Calder MD, Caveney A, Westhusin ME, Watson AJ. 2000. Expression of cyclooxygenase -2(COX-2) and prostaglandin E receptors 2 and 3 (EP2 and EP3) during maturation in vitro of bovine cumulus oocyte complexes (COCs). Biol. Reprod. 62:271. Abstract.
- King WA, Betts DH, Bordignon V, Hill JR, Winger QA, Westhusin ME, Smith LC. Assessment of telomerase activity and telomere length in cloned bovine embryos, fetuses and offspring derived by somatic cell nuclear transfer. Utah State University Genetically Engineered and Cloned Anima Symposium, June 17-20, 2000; Park City, Utah.
- Shin T, Sneed L, Hill JR, Westhusin ME. 2000. High incidence of developmental failure in bovine fetuses derived by cloning BVDV (Bovine viral diarrhea virus) infected cells. Therio, 53:243. Abstract.
- Hill JR, Long CR, Looney CR, Winger QA, Westhusin ME. 2000. Placental abnormalities in first trimester somatic cell cloned fetuses. Therio, 53:218. Abstract.
|
Progress 01/01/99 to 12/31/99
Outputs
The objective of this research program is to develop and improve methods for producing animals using assisted reproductive technologies. The primary focus involves the utilization if in vitro methods for producing mammalian embryos, and animal cloning. A large number of viable bovine embryos have been produced by nuclear transplantation (cloning). Some of these embryos were transferred into surrogate mothers to establish pregnancies. Fetuses produced by cloning were removed from surrogate mothers to determine if they were developing normally. Others were allowed to develop to term and one resulted in a viable bull calf cloned from a skin cell derived from a 21-year old Brahman bull. Results of these studies indicated that most embryos derived by cloning are abnormal and the fetuses die between day 30 and 80 of gestation. Abnormal development of the placenta seems to be the major cause of failed development. Gene expression in cloned fetuses is abnormal when compared
to normal controls. Thus far, studies have been targeted an analyzing expression of genes encoding IGF family members. Cloned fetuses appear to express lower levels of IGF II. Cell lines derived from an Angus bull believed to be naturally resistant to Brucellosis were also utilized for cloning. These cells were frozen and stored in liquid nitrogen in 1985. A number of viable embryos were produced using this cell line. Several pregnancies were obtained. Only one survived past 120 days of gestation but then spontaneously aborted at 7 months of gestation. Additional progress has been made with developing techniques for cloning canids. Procedures for collecting unfertilized oocytes to use for nuclear transfer have been developed. However, these involve surgical collections from live animals and the number of oocytes that can be collected is extremely limited. Methods for in vitro maturation of canine oocytes are being developed. Other studies are targeted at developing methods for
culturing canine embryos in vitro. Nuclear transplantation has been utilized in attempts to clone dogs, resulting in viable embryos; but no pregnancies have yet been established.
Impacts
Successful and repeatable techniques for cloning animals will have a tremendous impact and animal agriculture, animal and human health. Techniques can be used to preserve and then recover the genotype of valuable animals that are deceased. Cloning could also be utilized to replicate animals with superior genotypes for use in the production of meat and milk, and greatly improve the efficiency of genetic engineering in livestock and other animals.
Publications
- Natale, D., Westhusin, M.E. and Watson, A.J. 1999. alpha-Amanitin sensitive mRNA expression during bovine early development. Biol. Reprod. 60:159 (Abstract).
- Watson, A.J., Westhusin, M.E., De Sousa, P.A., Betts, D.H. and Barcroft, L.C. 1999. Gene expression regulating blastocyst formation. Theriogenology. 51(1):117-133.
- Westhusin, M.E., Hill, J.R., Winger, Q.A., Jones, K.L., DeSousa, P.A. and Watson, A.J. 1999. Reprogramming gene expression followingn uclear transfer into bovine oocytes. Biol. Reprod. 60:79 (Abstract).
- Winger, Q.A., Hill, J.R., Watson, A.J. and Westhusin, M.E. 1999. Characterization of a bovine cDNA encoding citrate synthase and expression during bovine pre-attachment development. Mol. Reprod. Develop. In press.
- Winger, Q.A., Hill, J.R., Watson, A.J. and Westhusin, M.E. 1999. Genetic reprogramming of phosphofuctokinase in nuclear transfer embryos produced using bovine fetal fibroblasts. Biol. Reprod. 60:268 (Abstract).
- Calder, M., Natale, D., Caveney, A., Barcroft, L.C., Westhusin, M.E. and Watson, A.J. 1999. Bovine oocyte maturation in vitro employing serum-free media. Biol. Reprod. 60:129 (Abstract).
- DeSousa, P.A., Winger, Q.A., Hill, J.R., Jones, K.L., Watson, A.J. and Westehusin, M.E. 1999. Reprogramming of fibroblast mRNA expression following nuclear transfer in bovine embryos. Cloning 1:63-69.
- Elhassan, Y.M., Kraemer, D.C. and Westhusin, M.E. 1999. A simple salt solution medium supplemented with yolk plasma and lactate (YPLM) supports development of preimplantation bovine embryos in vitro. Anim. Reprod. Sci. 57(3-4):153-166.
- Hill, J.R., Roussel, A.J., Cibelli, J.B., Edwards, J.F., Hooper, N.L., Miller, M.W., Thompson, J.A., Looney, C.R., Westhusin, M.E., Robl, J.M. and Stice, S.L. 1999. Clinical and pathologic features of cloned transgenic calves and fetuses (13 case studies). Theriogenology. 51(8):1451-1465.
- Hill, J.R., Winger, G.Q., Long, C.R., Looney, C.T., Thompson, J.A. and Westhusin, M.E. 1999. Development rates of bovine nuclear transfer embryos derived from adult and fetal cells. Biol. of Reproduction. In press.
- Hill, J.R., Winger, Q.A., Jones, K.L., Thompson, J.A., Burghardt, R.C. and Westhusin, M.E. 1999. Serum starvation of bovine fetal fibroblasts prior to nuclear transfer increases in vitro development rates. Therio 51:204 (Abstract).
- Lee, C.K., Moore, K., Scales, N., Westhusin, M., Newton, G. and Piedrahita, J.A. 1999. Isolation and genetic transformation of primoridial germ cells from cattle, goats, rabbits, and rats. Australasian J. Anim. Sci. In press.
|
Progress 01/01/98 to 12/31/98
Outputs
In collaboration with Dr. Andy Watson at the University of Western Ontario, experiments have continued to analyze the effect of in vitro culture environment on gene expression in bovine embryos. Data collected thus far clearly demonstrates that culture conditions can have a dramatic effect on the expression pattern of genes thought to be important for normal embryonic development. Other experiments were conducted to determine the effect of supplementing culture medium with amino acids (aa), on bovine embryo development in vitro. Amino acids were measured in bovine uterine and oviductal fluid (OF). Concentrations of aa in the bovine reproductive tract were shown to be significantly higher than those of standard culture medium. Increasing the concentration of aa in culture medium to mimic those found in OF did not result in improved embryo development in vitro, however, supplementation of aa at levels approximating 10% of that found in OF did result in a significant
increase in development to the blastocyst stage. Significant progress was also made this past year with producing genetically identical cattle by nuclear transplantation using fetal and adult cells (cloning). A number of pregnancies have now been established using nuclear transplant embryos derived from both cell types. Some pregnancies are ongoing; however, many have aborted around 40 days of gestation. Studies continue to try and determine the cause of this phenomenon. In another collaborative effort with Dr. Watson, patterns of gene expression were analyzed and compared between normal embryos and embryos produced by nuclear transplantation. Results of this work indicated that gene expression was very similar between different embryo types, however a few differences were detected between normal embryos and cloned embryos. Analysis of these differences is currently underway with the hope this will provide some clues to the abnormal development seen in clones. Experiments to test the
effect of serum starvation of fetal and adult cells prior to nuclear transplantation demonstrated that serum starvation of fetal cells resulted in improved development however there was no advantage to serum starvation of adult cells. A new method for oocyte activation involving treatment of ova with inomycin followed by butyrolactone was tested and shown to be an effective method for activating oocytes following nuclear transplantation.
Impacts
(N/A)
Publications
- Chapman S.A., Keller D.L., Westhusin M.E., Drew M.L., Kraemer D.C. 1999. In vitro production of Axis deer (Axis axis) embryos, a preliminary study. Therio. In press.
- Elhassan Y.M., Tasca R.J., Westhusin M.E. 1999. Levels of amino acids in bovine oviductal and uterine fluids in comparison to levels available in a culture medium supplemented with commercial stocks. Therio. In press.
- Keller D.L., Chapman S.A., Westhusin M.E., Drew M.L., Kraemer D.C. 1999. Production of in- vitro derived blackbuck antelope (Antilope cervicapra) embryos using standard bovine protocols. Therio. In press.
- Lee C.P., Elhassan Y.M., Westhusin M.E. 1999. The effect of mercaptoethanol and N-acetyl cysteine on development and hatching of bovine blastocysts produced in vitro. Therio. In press.
- Hill J.R., Winger Q.A., Jones K.L., Thompson J.A., Burghardt R.C., Westhusin M.E. 1999. Serum starvation of bovine fetal fibroblasts prior to nuclear transfer increases in vitro development rates. Therio. In press.
- Hill J.R., ., Winger Q.A., Jones K.L., Thompson J.A., Burghardt R.C., Westhusin M.E. 1999. Elucidation of factors affecting the in vitro development of embryos derived from bovine fetal fibroblasts. Proc. Theriogenology Ann. Mtg. In press.
- Winger Q.W., Elhassan Y.M., Watson A.J., Westhusin M.E. 1998. Expression of lactate dehydrogenase mRNAs in bovine pre-attachment embryos. Biol. Reprod. 58(Suppl 1):77.
- Natale D.R., Westhusin M.E., Watson A.J. 1998. Characterization of differentially expressed mRNAs in bovine pre-attachment embryos cultured in defined and serum-supplemented/co-culture systems. Therio. 49:188.
- Hill J., Westhusin M.E., Varner Dickson D. 1998. Advances in large-animal reproduction: In vitro production of embryos. Veterinary Medicine/June:575-582.
- Hill J., Westhusin M.E., Varner Dickson D. 1998. Advances in large-animal reproduction: The expanding role of ultrasonography. Veterinary Medicine/May:480-484.
- Hill J., Westhusin M.E., Varner Dickson D. 1998. Advances in large-animal reproduction: Updated techniques for artificial insemination and embryo transfer. Veterinary Medicine/May:485-490.
- Watson A.J., Westhusin M.E., Winger Q.A. 1998. IGF paracrine and autocrine interactions between conceptus and oviduct. J. Reprod. Fertil. In press.
- Azambuja R.M., Kraemer D.C., Westhusin M.E. 1998. Effect of low temperatures on in vitro matured bovine oocytes. Therio. 49:1155-1164.
- Westhusin M.E. 1997. From mightly mice to mighty cows. Nature Genetics News & Views 17:4-6.
|
Progress 01/01/97 to 12/31/97
Outputs
A number of experiments targeted at developing improved methods for culturing bovine embryos were conducted. Completely defined systems for culturing bovine embryos in vitro were established, however the addition of serum to culture medium still results in a higher proportion of the embryos developing to the blastocyst stage. Attempts to understand the beneficial effect of serum included experiments designed to investigate the effects of adding antioxidants to culture medium. Preliminary results indicate a beneficial effect of antioxidants, however they can not be used to completely replace serum supplementation. In collaboration with Dr. Andy Watson at the University of Western Ontario, techniques were established to analyze gene expression in single bovine embryos and oocytes. High standard errors obtained when analyzing single embryos suggested that analyzing pools of embryos may provide a more accurate approach, however, this eliminates the ability to study
individual variation between samples. Irregardless, data from additional experiments clearly demonstrated that gene expression profiles of bovine embryos produced in vitro were different than those collected from naturally mated cows. Also, in vitro culture environment effected the pattern of gene expression. In collaboration with Dr. Jorge Piedrahita, experiments were initiated to produce transgenic cattle by cloning genetically engineered fetal cell lines.
Impacts
(N/A)
Publications
- ESTRADA, J.L., PACHON, L.A., OLIVERA, M., PIEDRAHITA, J., and WESTHUSIN, M. 1998. Superovulatory response of Colombian Creole cattle to two doses of FSH. Proc. International Embryo Transfer Society. Theriogenology, in press. (abstract).
- CARMARGO, D.O., WESTHUSIN, M., ESTRADA, J.L., GALLEGO, M.I., OLIVERA-ANGEL, M. 1998. Effects of superoxide dismutase and thioredoxin on bovine embryo development in vitro. Proc. International Embryo Transfer Society. Theriogenology, in press. (abstract).
- ELHASSAN, Y.M. and WESTHUSIN, M.E. 1997. An egg-yolk fraction high in LDL supports in vitro development of bovine embryos in a cell-free basic salt solution supplemented with lactate. Biol. Reprod. 56 (Suppl 1):138. (abstract).
- ELHASSAN, Y.M. and WESTHUSIN, M.E. 1997. In vitro development of preimplantation bovine embryos in a coculture system supplemented with egg yolk. Theriogenology 47:288. (abstract).
- DeSOUSA, P.A., WESTHUSIN, M.E. and WATSON, A.J. 1997. Analysis of variation in relative mRNA abundance for specific gene transcripts in single bovine oocytes and early embryos. Mol. Reprod. Dev. 49:119-130.
- DeSOUSA, P.A., CAVENEY, A., WESTHUSIN, M.E. and WATSON, A.J. 1997. Analysis of temporal patterns of zygotic gene expression and the maternal factors which regulate them. Theriogenology, in press. (abstract).
- AZAMBUJA, R.M., KRAEMER, D.C. and WESTHUSIN, M.E. 1997. Effect of low temperatures on in vitro matured bovine oocytes. Theriogenology, in press. (abstract).
- AZAMBUJA, R.M., KRAEMER, D.C. and WESTHUSIN, M.E. 1997. Effects of low temperatures on in vitro produced bovine zygotes. Mol. Reprod. Dev. 47:435-439.
|
Progress 01/01/96 to 12/30/96
Outputs
Successful methods for culturing bovine embryos to the blastocyst stage without co-culture with somatic cells were developed. Experiments designed to evaluate the effect of adding serum or egg yolk to culture medium were conducted. Data from these experiments indicated a beneficial effect with more embryos developing to the blastocyst stage when either serum or egg yolk were added to culture medium. Initial experiments to test the effect of in vitro embryo culture conditions on gene expression were carried out; however, data collected to this point are inconclusive. Experiments designed to develop procedures for producing viable bovine embryos by injecting single non-motile sperm into unfertilized oocytes were conducted. Problems with egg lysis following sperm injection were overcome by the addition of 20% fetal calf serum to the manipulation medium and improvement in our technical skills. Fertilization rates following sperm injection as indicated by the presence of 2
pronuclei were higher in ova that were artificially activated following sperm injection but still lower than in control oocytes fertilized in vitro (IVF) with motile sperm.
Impacts
(N/A)
Publications
- WESTHUSIN, M. E., ET. AL. 1996. Effects of low temperatures on in vitro produced bovine zygotes. Mol. Reprod. Dev. (Accepted).
- WESTHUSIN, M. E., ET. AL. 1996. In vitro maturation of ovine oocytes in the absence of continuous 5% CO2 in a portable incubator. Therio. In press.
- WESTHUSIN, M. E., ET. AL. 1996. Antibodies to SP-10 inhibit bovine fert. in vitro. J. Reprod. Fert. 107:287-297.
- ELHASSAN, Y.M. and WESTHUSIN, M.E. 1997. In vitro development of preimplantationbovine embryos in a coculture system supplemented with egg yolk. Therio. (accepted).
- ELHASSAN, Y.M. and WESTHUSIN, M.E. 1996. In vitro development of bovine embryos...in the presence/absence of fetal bovine serum and bovine serum albumin. Biol. Reprod. 54(Suppl 1):81.
- JONES, K.L. and WESTHUSIN, M.E. 1996. Effect of polyvinyl alcohol, bovine serum albumin fraction V, fetal calf serum, and fetal calf serum plus bovine serum albumin fraction V on bovine embryo development. Therio. 45(1):205.
- WESTHUSIN, M.E. and AZAMBUJA, R.M. 1996. Development of in vitro-derived bovine embryos following pronuclear transplantation and in vitro culture. Anim. Reprod.Sci. 45:29-35.
|
Progress 01/01/95 to 12/30/95
Outputs
Experiments were conducted to compare different methods for culturing bovine embryos in vitro. Co-culture of embryos with BRL cells, in CR1aa medium supplemented with fetal calf serum resulted in the highest percentage of embryos developing to the blastocyst stage when compared to embryo culture in medium alone and/or without serum supplementation. Only those embryos cultured with BRL cells survived cryopreservation. Preliminary experiments to test the effect of egg yolk on in vitro embryo culture were also conducted. Results of these experiments were very encouraging in that the addition of 5% egg yolk to culture medium nearly doubled the percentage of embryos developing to the blastocyst stage. Experiments designed to develop procedures for producing viable bovine embryos by injecting single non- motile sperm into unfertilized oocytes were conducted. Although fertilization was obtained using this procedure, the percentage of eggs exhibiting normal fertilization
following sperm injection was less than 10%. In addition, the great majority of the eggs did not survive the injection procedure. Experiments are underway to investigate new approaches which will allow a higher percentage of the eggs to survive post-injection. Viable bovine embryos were produced following manipulation of cytoplasmic inheritance using pronucleus transplantation. Mitochondrial DNA was sequenced in 4 donor cows which will be used for ongoing studies involving the manipulation of cytoplasmic inheritance in cattle.
Impacts
(N/A)
Publications
- JONES KL & WESTHUSIN ME. 1996. Effect of polyvinyl alcohol, bovine serum albumin fraction V, fetal calf serum, and fetal calf serum plus bovine serum albumin fraction V on bovine . . . Therio. (Accepted).
- WESTHUSIN ME & PIEDRAHITA JA. 1995. Application of animal biotechnology to the beef industry. Korean J. Emb. Trans. 10:1-13.
- SCHULTZ GA, et al., 1995. Regulation of early embryonic development by growth factors: Growth factor gene expression in cloned bovine embryos. J. Anim. Sci. (in-press).
- WESTHUSIN ME, et al., 1995. Reducing . . . cytoplasm available . . . embryonic development decreases the quality . . . quantity of embryos . . . in vitro fertiliz. & nuclear transplant. Therio. (in-press).
- WESTHUSIN ME & AZAMBUJA RM. 1995. Development of in vitro-derived bovine embryos following pronuclear transplantation and in vitro culture. Anim. Reprod. Sci. (Accepted with minor revisions).
- WESTHUSIN ME, et al., 1995. Nuclear transfer in the bovine embryo: A comparison of different methods . . . . Therio. (In revision).
- DAY WE, et al., 1995. Characterization of the cervix in cycling mares using ultrasound. Biol. Reprod. Monograph 1, pp 15.
|
Progress 01/01/94 to 12/30/94
Outputs
Experiments were conducted to compare different methods for culturing bovine embryos in vitro. Co-culture of embryos with oviductal cells resulted in a higher percentage developing to the morula or blastocyst stage than did culture in medium alone. Successful methods for in vitro maturation of sheep oocytes in a portable incubator were developed. This allows for oocytes to be collected at distant locations then shipped to the laboratory overnight for fertilization and culture in vitro. Gene expression was analyzed in three types of bovine embryos: 1) embryos collected from cows, 2) embryos produced in vitro, and 3) embryos produced by nuclear transplantation. Reverse transcription polymerase chain reaction was used for the initial studies and indicated that patterns of gene expression were similar between embryos produced in vitro and in vivo, but embryos produced by nuclear transplantation were expressing higher levels of IGF-II, IGF-I receptor, TGF(alpha), and bFGF,
and lower levels of IGF-I. An alternative technique for analyzing gene expression in embryos was developed using reverse transcription and antisense RNA amplification. The primary advantage of this technique is that expression of a large numbers of different genes can be analyzed simultaneously. Techniques were developed to manipulate cytoplasmic inheritance in cattle using pronuclear transplantation. Pronuclear transplantation was then utilized to show that the cytoplasm of 1-cell bovine embryos is more sensitive to cool temperatures than is the nucleus.
Impacts
(N/A)
Publications
|
|