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

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

Understanding Developmental Abnormalities in Offspring Produced by Nuclear Transplantation

Role of Cell Cycle

Cloning and Other Reproductive Technologies for Application in Transgenics

HOMOLOGOUS RECOMBINATION AND GENETIC ENGINEERING OF TRANSGENIC RECOMBINANT ANIMALS

Participants:

Sushma Pati, Geoff Sargent and Mary E. Steven
Pangene Corporation

Homologous recombination (HR) is used to manipulate chromosomal DNA in mouse embryonic stem (ES) cells to produce transgenic mouse models or in cell lines to study gene function by disrupting the natural function of a specific desired target gene. Using currently available standard HR methods, generally only about one in 100,000 to 10,000,000 target cells homologous recombines with the added DNA. In contrast, typically 1 in 1,000 cells randomly integrates the added DNA into the chromosome. Several drug selection protocols are available to enrich for rare homologous recombinants in the DNA transfected cell populations. However, the recombinant cells' selective growth advantage afforded by these drug selection approaches is often limited. This means one must screen many cells to find the desired homologous recombinants. As a consequence, identifying the homologous recombinants in a large population of non-homologous random integrants is often the rate limiting step for creating homologously modified mammalian cells. This severely limits the ability to systematically manipulate target genes. Furthermore, with existing drug selection strategies, the initial gene modification often leaves behind the drug selectable marker gene, unless additional rounds of gene targeting are performed. These strategies are labor intensive, time consuming, and ultimately limits HR genetic engineering of mammalian cells for commercial applications.

Although there are intensive efforts to isolate and culture ES cells which can contribute to the germline of large animals, currently ES cells from only a few strains of mice are available. For strains of mice where ES cells have not been isolated and other animals, only random transgene additions are now commercially feasible. Thus, HR has currently not been enabled for large animal transgenic production.

Routine methods for producing non-mouse transgenic animals, including rats, rabbits, pigs, goats, or cattle, generally rely on direct microinjection of DNA encoding a transgene into the pronucleus of fertilized zygotes. Current classical pronuclear microinjection methods result in the random integration of transgenes at one or more locations in the chromosome of the zygote. It is not possible to predict expression levels of the transgene and thus extensive, expensive characterizations must be performed for every transgenic experiment to derive a line of animals that stably express the desired transgene at appropriate levels. To circumvent unpredictable protein expression levels from randomly integrated genes, it is often advantageous to include large regions of flanking DNA in the transgene construct in order to ensure position independent transgene expression. While large transgenes have been used in the generation of transgenic animals, transgene integration frequencies are often far lower than with cDNA constructs.

A third method for producing transgenic animals relies on the use of nuclear transplantation methods, which transfers the complete genetic material (the DNA contained in a donor nucleus) from a donor cell into a recipient egg cell, whose own nucleus is removed. Following nuclear transfer and activation, recipient cells can form a viable embryo which then develops into a normal animal. The donor cells that provide the nuclear genetic material can be differentiated somatic cells from fetal fibroblasts, adult mammary epithelial cells, or other cells derived from embryos. The donor cell is grown in culture and can be genetically modified with a desired transgene prior to nuclear transplantation. These genetically modified cells are characterized prior to nuclear transplantation. With this approach, the expression levels of the desired transgene are defined prior to the nuclear transplantation step. The ability to use homologous gene recombination to modify the cell nucleus can greatly advance this method, enabling both gene deletions and modifications, in contrast to only random transgene additions. As discussed, current methods are relatively inefficient and limited by the requirement for drug selection to identify recombinant cells. Drug selection requires that cells must be cultured for many passages creating significant technical hurdles for efficient and viable nuclear transplantation.