Generation of PLTP transgenic rabbits

The transgenic rabbits expressing PLTP in the mammary gland were obtained by the conventional technique of microinjection⁴².

The transgenesis vector containing the sequence encoding recombinant human PLTP was digested with NotI restriction enzyme and the insert containing the transgene was isolated on agarose gel and then purified on ElutipD (Schleicher-Schuell, Ecquevilly, France) according to the manufacturer’s instructions, ethanol precipitated and then taken up in 10 mM Tris-HCl buffer, 0.1 mM EDTA, pH 7.4.

Embryo donor New Zealand rabbits, aged 20–24 weeks, were treated with subcutaneous injections of swine FSH/LH (Follicle Stimulating Hormone/Luteinizing Hormone, Stimufol®, REPROBIOL) for 3 days to stimulate follicle development. On the third day, the rabbit does were mated with male New Zealand rabbits and immediately after mating were given an intramuscular injection of Receptal® (Busereline acetate - synthetic luteinizing hormone - Intervet International B.V.). The recipient rabbits were aged 20–34 weeks. A synchronized pseudopregnancy was induced by an intramuscular injection of Receptal®.

On the 4th day, 18–19 h after mating, embryos were collected from the rabbit donors for the DNA microinjection procedure: DNA microinjection was carried out immediately after collecting the embryos (19–21 h after mating). The single-cell stage embryos were placed in a drop of medium under an inverted microscope equipped with Normarsky objectives and Narishige micromanipulators. Individual embryos were positioned and secured using a holding pipette. The transgene diluted to a concentration varying from l ng/µl to 6 ng/µl, in buffer containing 10 mM Tris-HCl, 0.1 mM EDTA, pH 7.4, was preferentially micro-injected into the male pronucleus of the embryo using an injection pipette.

Following the microinjection, the embryos were maintained in in vitro culture for 1 to 3 hours (37 °C, 5% CO₂ in air). The quality of the micro-injected embryos was then quickly assessed under a stereomicroscope. The intact embryos were then reimplanted under general anesthesia into the lumen of the oviducts of the synchronized recipient rabbits (10–17 embryos in each oviduct), using a surgical procedure (oviducts were exteriorized by laparotomy). Parturition mostly occurred naturally 29–31 days after embryo transfer. When necessary, it was triggered by injecting Ocytovem® (Ceva) on the 31st day. The ratio of young rabbits born compared with the number of embryos reimplanted ranged from 5 to 20%.

During embryogenesis, the microinjected recombinant DNA was randomly integrated into the genome. The newborn rabbits (10 days) were tested for the presence of the transgene by a biopsy of the ear. Genomic DNA was extracted and PCR (Polymerase Chain Reaction) was performed using specific primers for the recombinant insert. Rabbits for which the transgene was detected were called “Founders F0”. The founder F0 lines were further characterized by i) analysis of the number of transgene copies integrated in their genome, and ii) determination of the number of integration sites. The number of copies of the transgene integrated into the genome of each founder F0 line was determined by quantitative PCR and Southern blotting. This number varied, depending on the line, from 1 copy per cell to a hundred copies per cell. The number of integration sites was also determined by Southern blotting. This number varied, depending on the line, from1 to 3 locations per genome.