From Cell to Production

Technical Challenges of Cloning Pigs for BioMedical Research

Somatic Cell Nuclear Transfer in Mammals

SATACs and Transgenesis

Concerns About Gene Transfer and Nuclear Transfer in Domestic Animals

Prospects and Hurdles in Optimizing the Vascular Support of Engineered Tissues

ES Cells Make Neurons in a Dish

Nuclear Transfer and Gene Targeting in Domestic Animals: Bioreactors of the Future

Application of Nuclear Transfer Technology in the Generation of Pigs for Xenetransplantation

Genomics: Delivering Cell Culture Systems for Tissue Therapy

Nuclear Transfer Technology

Gene Targeting in Domestic Species: Challenges and Opportunities

Homologous Recombination and Genetic Engineering of Transgenic Recombinant Animals

Nuclear Transplantation in the Cow: Future Challenges

Enhancing Transgenics through Cloning

ES Cells Offer is a Power Tool for Understanding the Genetic Control of Tissue Development and for Screening Potential Therapeutic Drugs

Human Germline Engineering -- The Prospects for Commercial Development

Mammalian Artificial Chromosomes for Animal Transgenesis

Role of Cell Cycle

Cloning and Other Reproductive Technologies for Application in Transgenics

UNDERSTANDING DEVELOPMENTAL ABNORMALITIES IN OFFSPRING PRODUCED BY NUCLEAR TRANSPLANTATION

Participant:

Mark E. Westhusin
Dept. of Veterinary Physiology and Pharmacology
College of Veterinary Medicine
Texas A&M University
College Station, TX 77843-4466

It is now well established that a significant proportion of bovine offspring produced by nuclear transplantation (cloning) exhibit abnormal phenotypes at birth. When preimplantation-stage embryos are utilized as nuclei donors, the primary abnormality appears to be high birth weights , and the physiological problems associated with this which lead to low survival rate. It is as yet unclear whether calves produced when using fetal or adult cells for nuclear transplantation also exhibit increased birth weights; the number of offspring born to date is too small to allow for a definitive answer. However, a number of other abnormalities have been observed and many times lead to death at the time of birth or shortly thereafter. These include abnormal placentation, pulmonary hypertension leading to insufficient pulmonary perfusion and respiratory distress syndrome, enlarged/dilated right ventricles and patent ductus arteriosus. Besides the developmental abnormalities observed at the time of birth, it is clear that embryos produced by nuclear transplantation also result in decreased pregnancy rates following embryo transfer and fetal losses (abortions/resorbtions) are significantly higher.

The cause of abnormal embryo/fetal development remains unknown. Increased birth weights in both sheep and calves have previously been attributed to in vitro culture conditions. This suggests that developmental abnormalities observed in animals produced by nuclear transplantation may be caused by culture environment and not the nuclear transfer procedure itself. While culture environment may contribute, it is unlikely to be the major factor involved as developmental aberrations are much more common in animals produced by nuclear transplantation compared to those produced using standard procedures for in vitro oocyte maturation, in vitro fertilization, and in vitro embryo culture.

It is imperative that research be conducted to try and understand the cause for developmental abnormalities in cloned animals so to develop strategies for overcoming this problem. While the urgency of this may not seem so apparent in cases involving the production of transgenic animals where only a few animals are needed, it is obviously essential if large scale agricultural applications are ever to be realized. Moreover, even in the case of transgenic animals where only a few offspring are required, it is as yet unclear whether developmental abnormalities might manifest themselves later in life. There is some evidence in mice (Eppig et al) that this might be expected. A few valuable transgenic animals which appear fine at birth are not going to be worth much if they drop over dead as adults and prior to their usefulness being realized.

In our laboratory we have initiated preliminary attempts to try and understand the abnormal development observed in embryos produced by nuclear transplantation by analyzing gene expression patterns in early embryos and comparing them to embryos produced by in vitro methods or collected from cows (in vivo). To date most of our efforts have been concentrated on evaluating gene expression in blastocysts. Our observations indicate that differences in gene expression profiles do exist between embryos produced in vivo vs in vitro vs by nuclear transplantation. However, when just looking at differences in blastocysts, gene expression patterns are highly conserved. Analysis of over 1000 bands generated by using differential display polymerase chain reaction (DD-RT-PCR) indicate a difference of only 2% - 5% in bands that are either missing or expressed when different embryo types are compared. The significance of this observation is yet to be determined. Also, it is important to ascertain whether this small percentage of differences holds true when embryos at different stages of development are analyzed and compared. It may be the case that while the percentage of differences is small, these differences are critical and can go a long way to explain developmental abnormalities. It is our hope that this is the case, therefore, we are currently cloning and sequencing genes that are differentially expressed. We have now determined the sequence of several genes, however, so far only one gene has been identified with significant homology to IGF-I. This gene appears to be expressed in embryos produced in vivo and in vitro but not in cloned embryos. Results of previous work conducted in our laboratory using RT-PCR also suggested IGF-I was not expressed in blastocysts produced by nuclear transplantation.