GENERATION OF TRANSGENIC TURKEYS BY TRANSFECTION OF PRIMORDIAL GERM CELLS

Authors: WONG, E - VIRGINIA POLYTECHNIC INST; WENTWORTH, A - UNIVERSITY OF WISCONSIN; WENTWORTH, B - UNIVERSITY OF WISCONSIN; Proudman, John; EL HALAWANI, M - UNIVERSITY OF MINNESOTA

Submitted to: Transgenic Animal in Agriculture
Publication Type: Review Article
Publication Acceptance Date: 5/20/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary: The production of transgenic animals offers substantial utility in agriculture for the improvement of productive traits and the enhancement of nutrient properties. Transgenic poultry will have unparalled product usefulness and will also provide enormous research potential for studying avian biological development, as well as endocrinological, cellular and molecular mechanisms of avian growth, reproduction and immunity. However, the technology to produce transgenic poultry lags behind that of transgenic mammals. Distinct features of the avian reproductive system necessitate original techniques to introduce exogenous DNA into the genome of the bird. This review describes the transfection of turkey gonadal primordial germ cells (PGCs) with a fragment of the turkey prolactin gene oriented for prolactin antisense RNA synthesis. The transgene was detected in the injected founder animals as well as their subsequent transgenic progeny. In none line, the transgene has persisted for three generations. Surprisingly, DNA analysis in these transgenic offspring has shown that the transgene was probably not integrated into the genome, but rather was inherited as a stable episome. Other methods of gene transfer in the avian species, and the implications of transgenic research, are also discussed.

Technical Abstract: Gonadal Primordial Germ Cells (gPGCs) were cultured with gonadal somatic cells from the indifferent gonads of Stage 28 turkey embryos. Cultured gonadal PGCs and somatic cells were transfected with a Lipofectin:DNA complex. The plasmid DNA (ASVLTR-asPRL) contained the avian sarcoma virus long terminal repeat promoter and a fragment of the turkey prolactin gene, oriented for prolactin antisense RNA synthesis. Transfected cells were injected into the blood vascular system of Stage 15-17 turkey embryos. The gonads of F0 turkey embryos were screened for the presence of the plasmid. Twenty percent (3/15) of the pooled gonadal samples, representing 98 gonads, were positive by PCR. In subsequent generations, the presence of the transgene was screened by PCR and verified by genomic Southern blotting. In one pedigree (line 168) the transgene has persisted into the F3 generation, which demonstrates successful germline transmission. Surprisingly, the Southern blot patterns of these positive birds are consistent with the transgene existing as an episome, even though there is no known eukaryotic origin of replication in plasmid ASVLTR-asPRL. To verify the episomal nature of the transgene, total genomic DNA from three Southern blot positive F2 birds was used to transform E. coli . The recovered plasmids were shown to be identical to the original plasmid used for transfection by restriction enzyme mapping and DNA sequencing. In a second pedigree (line 9), the episomal transgene was unstable. It was detectable in the F2 generation but was lost in the F3 generation. These results demonstrate that transfection of a mixed population of gonadal PGCs and gonadal somatic cells can be used as an efficient method of generating transgenic turkeys.