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Positive selection of transgenic bovine embryos in culture
Thenogenology
POSITIVE
345
SELECTION
Gene Evaluation
OF TRANSGENIC
BOVINE
EMBRYOS
K_ R. Bondioli and R. J. Wall and Mapping Laboratory, USDA, Beltsville,
IN CULTURE
MD 20705 USA
A major constraint for the production of transgenic cattle is the cost of transfening embryos to recipients which do not produce transgenic cattle. Attempts to select transgenic embryos prior to transfer by biopsy and assay for the transgene have not been very efficient. We chose a non invasive approach for selecting transgenic embryos during culture. To accomplish this we constructed a transgene which included the neomycin resistance gene (neo) driven by a promoter known to be active in preimplantation embryos. The transgene contained a 3.2 kb segment of the chicken p actin promoter, the /3-galactosidase (P-Gal) coding region ofthe lac z gene with the nuclear locating signal of large T antigen of SV40, an internal riiosome entry site and the neo coding region. This construct produces a bi-cistronic mRNA allowing for translation of the lac z gene product and cap independent translation of the neo gene product. Oocytes were aspirated fi-om ovaries of slaughtered cows, matured in Ham’s F IO medium supplemented with 10% fetal calf serum (FCS) and 0.3 &ml LH for 22 to 24 h then inseminated with a sperm suspension containing 2 pg of heparin and approximately 1.5 x106 sperm per ml At 22 to 24 h after insemination cumulus cells were removed and pronuclei of centrifitged zygotes microinjected with the lac z / neo construct described above (3 ng DNA/pi). All cultures were carried out in CRlaa medium supplemented with 10% FCS, 10 mM glycine and 1 mhl alanine over neomycin (G418) resistant BRL cells at 50-60% contluency. Non injected zygotes were cultured as described above in the presence of 0, 25, 50 or 100 pg/ml G418. These treatments resulted in 40, 19, 9 and I %, respectively, of zygotes developing to the blastocyst stage after 8 days of culture. A total of 683 zygotes were microinjected and placed into culture (without G418) of which 354 (52%) cleaved. At 48 h post injection 138 and 160 embryos with greater than 2 cells were assigned to the control (medium alone) or G418 treatments (100 pg/ml ofmedium), respectively. The control treatment resulted in 26 (19%) Day 9 (insemination equals Day 0) blastocysts while those cultured in the presence of G418 resulted in 15 (9%) blastocysts. Blastocysts were stained for P-Gal activity, counter stained with Hoechst 33342 and mounted on slides. The number of cells with P-Gal activity and the total number ofnuclei were determined for each blastocyst. Proportions of cells stained for P-Gal activity were tested by Fisher’s exact test for 2 x 2 tables. Blastocysts in the control treatment had a mean of 88.3 cells ofwhich 24.5 (28%) stained for P-Gal activity. Blastocysts in the G418 treatment had a mean of 55.3 cells of which 45.7 (83%) stained for P-Gal activity (P c.05). In the control treatment 2 1 of 26 (81%) blastocysts had some cells with P-Gal activity while all of the blastocysts in the G4 18 treatment had some cells with P-Gal activity (P > .05). Nine of 15 (60%) blastocysts in the G418 treatment had greater than 80% cells with P-Gal activity while only 4 of 26 (15%) of those in the control treatment had greater than 80% cells staining for P-Gal (P < .05). These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G4 18. Further studies are anticipated to determine the viability of these embryos and if they will result in a higher proportion of transgenic fetuses.
POSITIVE
345
SELECTION
Gene Evaluation
OF TRANSGENIC
BOVINE
EMBRYOS
K_ R. Bondioli and R. J. Wall and Mapping Laboratory, USDA, Beltsville,
IN CULTURE
MD 20705 USA
A major constraint for the production of transgenic cattle is the cost of transfening embryos to recipients which do not produce transgenic cattle. Attempts to select transgenic embryos prior to transfer by biopsy and assay for the transgene have not been very efficient. We chose a non invasive approach for selecting transgenic embryos during culture. To accomplish this we constructed a transgene which included the neomycin resistance gene (neo) driven by a promoter known to be active in preimplantation embryos. The transgene contained a 3.2 kb segment of the chicken p actin promoter, the /3-galactosidase (P-Gal) coding region ofthe lac z gene with the nuclear locating signal of large T antigen of SV40, an internal riiosome entry site and the neo coding region. This construct produces a bi-cistronic mRNA allowing for translation of the lac z gene product and cap independent translation of the neo gene product. Oocytes were aspirated fi-om ovaries of slaughtered cows, matured in Ham’s F IO medium supplemented with 10% fetal calf serum (FCS) and 0.3 &ml LH for 22 to 24 h then inseminated with a sperm suspension containing 2 pg of heparin and approximately 1.5 x106 sperm per ml At 22 to 24 h after insemination cumulus cells were removed and pronuclei of centrifitged zygotes microinjected with the lac z / neo construct described above (3 ng DNA/pi). All cultures were carried out in CRlaa medium supplemented with 10% FCS, 10 mM glycine and 1 mhl alanine over neomycin (G418) resistant BRL cells at 50-60% contluency. Non injected zygotes were cultured as described above in the presence of 0, 25, 50 or 100 pg/ml G418. These treatments resulted in 40, 19, 9 and I %, respectively, of zygotes developing to the blastocyst stage after 8 days of culture. A total of 683 zygotes were microinjected and placed into culture (without G418) of which 354 (52%) cleaved. At 48 h post injection 138 and 160 embryos with greater than 2 cells were assigned to the control (medium alone) or G418 treatments (100 pg/ml ofmedium), respectively. The control treatment resulted in 26 (19%) Day 9 (insemination equals Day 0) blastocysts while those cultured in the presence of G418 resulted in 15 (9%) blastocysts. Blastocysts were stained for P-Gal activity, counter stained with Hoechst 33342 and mounted on slides. The number of cells with P-Gal activity and the total number ofnuclei were determined for each blastocyst. Proportions of cells stained for P-Gal activity were tested by Fisher’s exact test for 2 x 2 tables. Blastocysts in the control treatment had a mean of 88.3 cells ofwhich 24.5 (28%) stained for P-Gal activity. Blastocysts in the G418 treatment had a mean of 55.3 cells of which 45.7 (83%) stained for P-Gal activity (P c.05). In the control treatment 2 1 of 26 (81%) blastocysts had some cells with P-Gal activity while all of the blastocysts in the G4 18 treatment had some cells with P-Gal activity (P > .05). Nine of 15 (60%) blastocysts in the G418 treatment had greater than 80% cells with P-Gal activity while only 4 of 26 (15%) of those in the control treatment had greater than 80% cells staining for P-Gal (P < .05). These results clearly show an enrichment of blastocysts expressing the transgene in the majority of their cells after culture in the presence of G4 18. Further studies are anticipated to determine the viability of these embryos and if they will result in a higher proportion of transgenic fetuses.