Cassette DNA Fragment for Selection and Sexing of Preimplantation Bovine Transgenic Embryos

Abstract

A 369-bp cassette DNA fragment (LACFE) was constructed for simultaneous detection of exogenous gene integration and sexing of preimplantation bovine embryos. The LACFE consists of a part of the bovine lactoferrin gene, including three Dpn I sites and a pair of Y chromosome-specific primer sequences for polymerase chain reaction (PCR) of both ends. Bovine pronucleus embryos were microinjected with the LACFE modified by DNA adenine methylase. After 7 to 9 days culture in vitro, the blastocysts were bisected and the demi-embryos were analyzed by the PCR method following the nuclease treatment with Dpn I and Bal 31 nucleases (digestion-PCR) or without the nucleases (unmodified PCR), respectively. Results of sexing between a pair of demi-embryos agreed each other in 95 of 102 blastocysts. Ten of 102 demi-embryos were positive for the exogenous gene by unmodified PCR. On the other hand, 3 of 102 demi-embryos were positive by digestion-PCR, and included in the 10 demi-embryos. The digestion-PCR may exclude false-positive embryos, and prove to be an efficient selection of preimplantation bovine transgenic embryos. These results indicate that LACFE is useful for the simultaneous detection of the integration of exogenous genes and the sexing of preimplantation bovine embryos using only a single pair of primers. As the LACFE can be introduced into a construct of desirable transgene for microinjection, the LACFE is widely applicable for detection and sexing of transgenic embryos under the same analytical conditions.