Evaluation of Laser-Assisted Lentiviral Transgenesis in Bovine

Lentiviral transduction of oocytes or early embryos is an efficient
strategy to generate transgenic rodents and livestock. We evaluated
laser-based microdrilling (MD) of the zona pellucida, which is a
physical barrier for viral infection, and subsequent incubation in
virus suspension as a new route for lentiviral transgenesis in
bovine. Lentiviral vectors carrying an eGFP expression cassette
were used to transduce oocytes or zygotes after MD as compared to
the established subzonal virus injection technique (MI). The type
of manipulation (MD vs. MI) did not affect cleavage rates, but had
a significant effect on blastocyst rates (p < 0.001). MI of
virus or sham-MI (buffer) resulted in higher blastocyst rates as
compared to MD, both in the oocyte and zygote treatment groups. The
latter exhibited higher rates of early cleavage (p < 0.05) and
blastocyst rates (p < 0.01). The proportion of eGFP expressing
blastocysts was higher after infection of oocytes (MD: 44 ± 9%; MI:
67±8%) than after infection of zygotes (MD: 26 ± 8%; MI: 26 ± 9%).
Overall efficacy (eGFP-positive blastocysts per treated oocytes or
zygotes) was highest after MI of oocytes (18 ± 2%). Our study
demonstrates the feasibility of laser-assisted lentiviral gene
transfer into bovine oocytes and zygotes. However, further
optimization of the procedure is required, mainly to reduce the
incidence of polyspermy after MD of oocytes and to eliminate
negative effects of MD on early embryonic development.