Research

Determining the nature of the barrier to pregnancy

 Embryo manipulation can be simply removing an embryo from the donor animal and immediately transferring it to a surrogate mother, or it can be more complicated involving microsurgery on the embryo and maintaining the embryo in special culture systems before transferring the embryo to the surrogate mother.  Invasive procedures such as embryo splitting, blastocyst injection, pronuclear injection and nuclear transfer (cloning) understandably create circumstances that may perturb or derail embryo development; however, normal offspring often result.  One ongoing investigation has been how embryo manipulation adversely affects development and birth of calves.  Pregnancies after transfer of manipulated bovine embryos, in vitro fertilization, in vitro embryo culture, and embryo transfer experiments commonly result in the birth of calves with excessively high birth weights that may be associated with other abnormalities.  Together these symptoms are called Large Calf Syndrome.  Embryo, fetal, and placental growth and development seem to be affected even during the first few days after fertilization. We have made progress in describing the mechanisms to determine when during gestation abnormally high growth occurs (Bertolini web page).

Isolation of Embryonic Stem Cells from Embryos of Domestic Livestock:

Embryonic stem (ES) cells are undifferentiated cells that can be isolated and cultured from early mammalian embryos.  ES cells readily proliferate in vitro, can incorporate foreign DNA, and still retain their capacity to differentiate into normal tissues.  For example, a transgenic ES cell line can be created in vitro and the genetic modification be established in a living animal by combining the transgenic ES cells with a normally developing embryo.  The resulting animal can be a chimera that contains cells, including gametes, from both the ES cells and the host embryo.  The use of ES cells holds tremendous potential for modifying the genetic composition of domestic livestock, just as the power of using mouse ES cells in biomedical research is demonstrated daily throughout the world. 

 Our research efforts in this area are aimed at isolation of stem cells from porcine and bovine embryos.  Putative ES cells are lines are injected into blastocysts that are transferred to recipient for development to term.

We have established long-term cultures (Petkov web page) of primordial germ cells; the resulting cell lines are referred to as embryonic germ (EG) cells. Our EG cell lines have many characteristics in common with porcine ES-like cell lines isolated from blastocyst-stage embryos.  

Nuclear Transfer (Cloning) and Transgenic Animals:

Hans Spermann envisioned the process of cloning in 1938, but it took until 1997 to shock the world with the reality of cloning a mammal using an adult cell.  That shock came in the form of a sheep and her name was Dolly. The world of science and agriculture has not been the same since. 

Currently numerous species have been successfully cloned from adult cells worldwide.  The process of nuclear transfer (cloning) involves removing the nucleus, or genetic material from an egg and replacing it with the genetic material of a different cell.  The complex of enucleated egg and foreign genetic material is fused with a small charge of electricity and then allowed to develop in culture.  Embryos that result are transferred to the reproductive tract of a surrogate mother for development.  The process for cloning each species carries its own particular obstacles to overcome.  Cloning processes work but with very low efficiency due in part to a high incidence of embryonic and fetal loss.

A major area of uncertainty for successful nuclear transfer is the type of donor cell used to contribute genetic material to the cloned embryo (Batchelder’s web page).  Using a variety of cell types and different cattle breeds, several cloned calves have been produced in this laboratory.  All the pregnancies, including currently ongoing pregnancies, are monitored for growth and development. 

Cattle are the most commonly cloned livestock.  Their importance to agriculture makes this species a natural area of interest for research.  A project to scrutinize nuclear transfer bovine embryos at day-30 (Hoffert’s web page) seeks to define placental development and the events of implantation of early cloned embryos.  By describing gene expression, and the process of vascular growth we hope to describe the mechanisms to improve nuclear transfer in cattle.

Large offspring born from manipulated mammalian embryos have been described for most species studied.  The mouse provides a model to measure the contributions of fetal versus placental aberrations to abnormal development of cloned concepti (Mason’s web page) An exercise to combine cloned mouse embryos with in vivo-produced embryos subsequently transferred to surrogate mothers has been designed to show the respective influences of the embryo’s trophectoderm and inner cell mass (ICM).   

Comparative reproduction

Projects are also underway to examine reproductive processes in a range of species that includes large, laboratory and companion animals.  A long-term study is underway on the use of transgenic technology to alter milk composition in dairy cattle and goats.  Our collaborative efforts have involved several genes driven by a mammary gland-specific promoter.  Our results from studies in transgenic mice and goats have demonstrated that transgene expression is effectively targeted to the mammary gland (i.e., the transgene is expressed by mammary cells but not by other cells in the body.)  We have also demonstrated that transgene expression leads to altered physical and manufacturing properties of milk.