Lentiviral vectors have been shown to be a powerful tool to create transgenic livestock (Hofmann et al. 2003 EMBO Rep. 4, 1054-1060; Hofmann et al. 2004 Biol. Reprod. 71, 405-409). Due to their inability to pass the zona pellucida (ZP) by themselves, viral particles were delivered by subzonal microinjection of oocytes or zygotes. Here we show that an artificial opening of the ZP, generated by an infrared 1.48 �m microsurgical diode laser (OCTAX Laser Shot", kindly provided by MTG, Altdorf, Germany) and subsequent culture of oocytes in virus suspension, was sufficient to allow virus integration and expression of eGFP. The ZP of denuded in vitro-matured bovine oocytes was microdrilled with 2 to 4 laser shots of 3.0-ms pulse length each to create an opening of at least 40 �m. Oocytes were transferred to microdroplets (20 �L of Fert Talp) containing the virus suspension (lentiviral vector containing the eGFP reporter gene under the control of the human phosphoglycerate-kinase 1 promoter). After incubation for 2 or 4 h (5% CO2, maximum humidity, 39�C), oocytes were washed thoroughly in fresh droplets of medium to remove excessive virus. Sperm was added (0.5 � 106/mL) for 8 h. In vitro culture of embryos (5% CO2, 5% O2, maximum humidity, 39�C) was continued in culture medium (SOF) under oil for up to 8 days. Cleavage rate and number of blastocysts were determined on Days 2 and 8, respectively. Expression of eGFP was analyzed on Day 8 by fluorescence microscopy using an eGFP-specific filter with UV light source under an inverted microscope. In total, 267 oocytes were microdrilled. The range of optimal virus concentration was analyzed by titration. Seven different concentrations in a range of 1.25 � 106 to 5 � 107 IU/mL were used at an incubation time of 4 h. In one experiment using a high virus concentration, the incubation time was reduced to 2 h. Control groups were treated equally except for addition of virus. All experiments resulted in blastocysts showing eGFP expression in both the trophoblast and the inner cell mass. In four experiments we used aliquots of the same virus preparation (1.25 � 107 IU/mL) for infection. The cleavage rate ranged from 50 to 70%, and blastocyst rate from 15 to 35%. In three experiments showing good to average embryo development, 25 to 83% of the resulting blastocysts expressed eGFP, indicating efficient lentiviral transduction of oocytes following laser-assisted zona microdrilling. Studies directly comparing zona microdrilling and subzonal injection with regard to the efficacy of lentiviral transgenesis and the incidence of polyspermy are underway.
Restriction enzyme mediated integration and FIV lentiviral transgenesis applied to amphibians
