Progress 12/15/02 to 06/14/05
Outputs
Somatic cell nuclear transfer has excellent potential but is hindered by low efficiency due to the severe defects that afflict cloned animals throughout development. For nuclear transfer (NT) to have widespread commercial and biomedical applications, it is critical that research is done on gene expression and nuclear reprogramming in clones in order to understand and overcome the problems associated with this powerful technology. For the first time, we have examined global gene expression patterns in bovine preimplantation nuclear transfer, in vivo and in vitro fertilized bovine embryos utilizing cDNA microarray technology. We have shown that the expression profile of cloned embryos closely resembles that of their in vivo counterparts and is drastically different from the fibroblast donor cell. Furthermore, we examined genes involved in or affected by nuclear reprogramming (i.e. imprinted genes, X-linked genes, and genes responsible for epigenetic regulation and
chromatin modification) and found no differential expression between embryos generated by nuclear transfer or in vivo. Taken together, the evidence demonstrates that the NT embryos have undergone significant nuclear reprogramming by the blastocyst stage.
Impacts
We have shown that nuclear reprogramming is relatively complete at the blastocyst stage of cloned embryo develolpment, thus setting up the justification for studying clone abnormality to the fetal stage.
Publications
- Smith, S.L., Sung, L-Y., Page, R., Henderson, B., Du, F., Everts, R., Nedambale, T., Rodriguez-Zas, S., Renardm J-P., Lewin, H., Yang, X., Tian, X.C. 2006. Global Gene Expression Profiling of Single Bovine Embryos Reveals Significant Effects of In Vitro Maturation, Fertilization and Culture on Gene Expression. Reprod. Fert. Dev. 18:111
- Everts, R.E., Razzak, A., Chavatte-Palmer, P., Hue, I., Green, C.A., Oliveira, R., Rodriguez-Zas, S., Tian, X.C., Yang, X., Renard, J-P, Lewin, H.A. 2006. Global gene expression profiling distinguishes placentomes derived by artificial insemination from those pregnancies derived by either in vitro fertilization or nuclear transfer. PAG abstract guide: p282. Presented at the Plant & Animal Genome XIV Conference, January 14 - 18, 2006, San Diego, California
- Smith, S.L., Everts, R.E., Tian, X.C., Du, F., Sung, L-Y., Rodrigues-Zas, S., Jeong, B-S., Renard, J.P., Lewin, H.A., Yang, X. 2005. Global Gene Expression Profiles of Cloned Blastocysts Reveal Significant Nuclear Reprogramming. PNAS 2005;102:17582-7
- Everts, R.E., Chavatte-Palmer, P., Razzak, A.A., Hue, I., Rodriguez-Zas, S., Tian, X.C., Yang, X., Renard, J-P., Lewin, H.A. 2005. Global gene-expression profiling of cattle placentomes collected from term pregnancies derived by artificial insemination, in vitro fertilization and nuclear transfer. PAG abstract guide: p244. Presented at the Plant & Animal Genome XIII Conference, January 15 - 19, 2005, San Diego, California
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Progress 01/01/04 to 12/31/04
Outputs
Nuclear transfer (cloning) has potential applications in agriculture and biomedicine, but the technology is hindered by low efficiency. Global gene expression analysis of clones is important for the comprehensive study of nuclear reprogramming. In this project, we used a powerful 7,872 cDNA bovine microarray to compare global gene expression profiles of 15 individual bovine cloned blastocysts with their somatic nuclear donor cells and fertilized control embryos. The gene expression profiles of cloned embryos were drastically different from those of their nuclear donor cells and closely resembled those of the naturally fertilized embryos. In this study, we found an 84.2% difference in gene expression patterns between the nuclear donor cells and the cloned embryos. Furthermore, we found that the gene expression profiles of the cloned embryos closely resembled those of the naturally fertilized embryos, more so than the IVF embryos. Our findings demonstrate that the
cloned embryos have undergone significant nuclear reprogramming by the blastocyst stage; however, it is possible that problems occur during re-differentiation for tissue- and organogenesis and that small reprogramming errors may be magnified downstream in development.
Impacts
The identification of differentially expressed genes in cloned vs. in vivo produced embryos allows for the development of new strategies in improving the overall nuclear transfer efficiency.
Publications
- No publications reported this period
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Progress 01/01/03 to 12/31/03
Outputs
During the report period, we screened the animals of the university research farm and identified one animal that is heterozygous for all polymorphisms in the forms of single nucleotide polymorphisms that we have identified in cattle. These polymorphisms reside in the following imprinted genes: H19, Igf2, Igf2r, and Mao A. The reason that we decided to clone an animal that is heterozygous for multiple imprinted genes is that we would be able to study the expression of imprinted genes in these cloned fetuses and be able to compare the cloned animals with control animals for their imprinting patterns. Additionally, we have generated more than 200 cloned and vitrified embryos from this animal using our new cryopreservation method, which would give us more than 90% survival after thawing. We have also generated more than 40 in vivo embryos for this study as controls for the cloned embryos. Recipients of these embryos are ready for embryo transfer in our collaborators farm.
We will be conducting embryo transfer and collect tissues from fetuses generated from these pregnancies in the new reporting period.
Impacts
Imprinting abnormalities has been proposed to be the major cause for the developmental abnormalities in cloned animals. The use of an animal that is heterozygous for numerous imprinting genes for nuclear transfer is important in that this will give us the opportunity to generate sufficient information on the cause of clone developmental abnormalities.
Publications
- Savage, AF, J Maull, XC Tian, M Taneja, L Katz, M Darre and X Yang. Behavioral Observations of Adolescent Holstein Heifers Cloned from Adult Somatic Cells. Theriogenology 60:1097-1110. 2003.
- Enright, BP, BS Jeong, X Yang and XC Tian. Epigenetic Characteristics of in vitro Cultured Donor Cells for Nuclear Transfer: Levels of Histone Acetylation. Biol Reprod 69:1525-1530, 2003.
- Lee J, Wu S-C, Tian X, Barber M, Hoagland T, Riesen J and Yang X. Production of Cloned Pigs by Whole Cell Intracytoplasmic Microinjection. Biol Reprod 69:995-1001, 2003.
- Lee, J-W, X Cindy Tian and Xiangzhong Yang. Failure of male pronucleus formation is the major cause for the lack of fertilization and embryo development in pig ICSI oocytes. Biol Reprod 68:1341-1347. 2003.
- Enright, BP, C Kubota, X Yang and XC Tian. Epigenetic Characteristics of Donor Cells treated by Trichostatin A or 5-Aza-2-deoxycytidine and development of embryos cloned from treated donor cells. Biol Reprod 69:896-901, 2003.
- Tian XC, Kubota C, Enright B, Yang X. Cloning animals by somatic cell nuclear transfer-biological factors. Reprod Biol Endocrinol 1:98-104, 2003.
- Liu JL, Sung LY, Du FL, Julian M, Jiang S, Barber M, Xu J, Tian X and Yang X. Differential Development of Rabbit Embryos Derived from Parthenogenesis and Nuclear Transfer. Mol Reprod Dev (in press), 2004
- Nedambale TL, Dinnyes A, Groen W, Dobrinsky JR, Tian X and Yang X. Comparison on in vitro fertilized bovine embryos cultured in KSOM or SOF and cryopreserved by slow freezing or vitrification. Theriogenology (accepted), 2004
- Lee J, Tian X and Yang X. Optimization of Parthenogenetic Activation Protocol in Porcine. Mol Reprod Dev (in press). 2004
- Liu, J-L, Hirokazu Kusakabe, Ching-Chien Chang, Hiroyuki Suzuki, David W Schmidt, Marina Julian, Robert Pfeffer, Charles L Bormann, X. Cindy Tian, R. Yanagimachi, and X. Yang. Freeze-dried sperm fertilization led to term development in rabbits. Biol Reprod (accepted). 2004.
- Jiang L, Carter DB, Xu J, Yang X, Prather RS, Tian XC. Telomere Lengths in Cloned Transgenic Pigs. Biol Reprod (in press) 2004.
- Chang CC, Nagy P, Abdelmassih R, Yang X, Tian XC. Morphology of the G2/M nuclei during somatic cell haploidization by GV stage mouse oocytes. Biol Reprod (in press) 2004.
- Lee, J.W. 2003. Production of pig embryos following microinjection of somatic cells and sperm. Ph.D. thesis. University of Connecticut.
- Enright, B. 2003. Development and subsequent reproductive performance of cloned cattle derived from adult somatic cells. Ph. D. thesis. University of Connecticut.
- Kubota, C. 2003. Studies on the development of bovine somatic cell nuclear transfer and its application for the production of super sire bull. Ph. D. thesis. University of Kagoshima.
- He, J. 2003. Quantification of extracellular matrix gene expression in single bovine oocytes and preimplantation embryos derived from IVF using real time RT-PCR. MS thesis. University of Connecticut.
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