Ewerling Sonja, Hofmann Andreas, Klose Regina, Weppert Myriam, Brem Gottfried, Rink Klaus, Pfeifer Alexander, Wolf Eckhard
Institute of Molecular Animal Breeding/Gene Center, Ludwig-Maximilians University, Munich, Germany.
Transgenic Res. 2006 Aug;15(4):447-54. doi: 10.1007/s11248-006-0015-2.
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.
慢病毒转导卵母细胞或早期胚胎是产生转基因啮齿动物和家畜的有效策略。我们评估了基于激光的透明带微钻孔（MD），透明带是病毒感染的物理屏障，随后在病毒悬液中孵育作为牛慢病毒转基因的新途径。与已建立的透明带下病毒注射技术（MI）相比，携带eGFP表达盒的慢病毒载体用于MD后转导卵母细胞或受精卵。操作类型（MD与MI）不影响卵裂率，但对囊胚率有显著影响（P<0.001）。在卵母细胞和受精卵处理组中，病毒的MI或假MI（缓冲液）导致的囊胚率均高于MD。后者表现出更高的早期卵裂率（P<0.05）和囊胚率（P<0.01）。卵母细胞感染后表达eGFP的囊胚比例（MD：44±9%；MI：67±8%）高于受精卵感染后（MD：26±8%；MI：26±9%）。卵母细胞MI后总体效率（每个处理的卵母细胞或受精卵中eGFP阳性囊胚）最高（18±2%）。我们的研究证明了激光辅助慢病毒基因转移到牛卵母细胞和受精卵中的可行性。然而，需要进一步优化该程序，主要是为了降低卵母细胞MD后多精入卵的发生率，并消除MD对早期胚胎发育的负面影响。
