Selection Of Transgenic Embryos Research Articles

Using GFP as a Scorable Marker in Walnut Somatic Embryo Transformation

Somatic embryogenesis is the foundation of genetic transformation in several economically important tree species. In the Juglans regia L. (Persian walnut) somatic embryogenesis-based transformation system, a major limiting factor is the selection of non-chimeric transgenic embryos in tissue culture. We transformed Persian walnut somatic embryos with the S65T synthetic green fluorescent protein (GFP) gene in order to assess the effect of this visual marker gene on embryo viability and the selection of transgenic embryos. Following a 10 d period of transient GFP expression in all inoculated embryos, stable fluorescent sectors were apparent in several embryos, allowing efficient and rapid visual selection of primary transgenic embryos. Two chimeric embryos were selected 40 d after transformation, and these two embryos gave rise to 13 stable transgenic embryo lines and 44 whole plants. GFP-expressing walnut plants and embryos developed normally and transformation was verified by GUS analysis. Our analysis suggests that the use of GFP as a selectable marker can significantly reduce labour, cost, and time in the walnut somatic embryogenesis-based transformation system.

Production of Bovine Transgenic Conceptus; Possible Selection of Transgenic Embryos by Polymerase Chain Reaction.

This study was conducted to examine the possibility of selection of transgenic bovine embryos by use of polymerase chain reaction (PCR) following microinjection of exogenous DNA before embryos were transferred to recipient cattle. DNA of bovine α-lactalbumin (α-LA) gene was microinjected into pronuclei of bovine embryos derived from in vitro maturation and fertilization. They were then cultured for 7 to 8 days to the blastocyst stage. To examine persistence of injected DNA during embryo development, embryos were analyzed by PCR at 0, 1, 3, 5 and 7 days following DNA microinjection. To distinguish the injected α-LA from the endogenous gene, gene-gene junction regions of head-to-tail concatemers of the injected DNA, which were formed when linearized DNA were injected into cell nuclei, were amplified by PCR. Injected DNA persisted at high rates (72 to 80%) until 5 days following DNA injection, but the detection rate at the blastocyst stage (20%) was lower than earlier stage (P<0.05), indicating that injected DNA into bovine embryos drastically decreased at the blastocyst stage. To determine if the PCR signals reflect transgenic status, biopsy samples of DNA-injected blastocysts were analyzed. Total of 1, 583 embryos were microinjected, and 124 (8.9%) developed to blastocysts. Twelve (11%) out of 109 samples biopsied from microinjected blastocysts were positive by PCR analysis. Eight PCR-positive embryos were transferred to 8 recipients. Also, 31 negative embryos were transferred to recipient heifers. Four out of 8 recipients that received PCR-positive embryos were pregnant and fetuses and placentae were recovered at 60 to 75 days of pregnancy. One of the samples, of which only a placenta was recovered, was found to be transgenic by both PCR analysis and Southern hybridization. The other samples had no injected DNA. On the other hand, 14 fetuses with placentae were recovered from recipients that received PCR-negative embryos. None of these samples were found to carry injected DNA. Our system could not detect single, or head-to-head- or tail-to-tail-joining transgenes; however, the results described here imply selective transfer of potential transgenic embryos to recipient heifers by the PCR selection.