Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos

Abstract

Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

Generation of RAG 1- and 2-deficient rabbits by embryo microinjection of TALENs

“…The efficiency of KO in founder offspring is extremely high, reaching 94% for RAG1 TALENs and 100% for RAG2 TALENs. These efficiencies are much higher than those observed from the production of random integration transgenic rabbits by pronuclear injection (< 10%) [10]. Moreover, the biallelic modification efficiency is very high (61% for RAG1; 100% for RAG2).…”

Hypodiploidy as a prominent attributor to chromosomal aneuploidy in transgenic rabbit embryos

“…19-20 h after the mating of hormonally treated donors (PMSG and HCG;Chrenek et al, 2005), the pronuclear stage eggs were flushed from the oviducts of the animals with PBS. The selection of flushed eggs was done in CIM medium + 10% FCS (Gibco BRL, USA).…”

“…The selection of flushed eggs was done in CIM medium + 10% FCS (Gibco BRL, USA). The embryos were cultured in in vitro conditions (5% CO 2 , 39°C, k-DMEM + 10% FCS; Gibco BRL, USA) as reported by Chrenek et al (2005).…”

“…The eggs were fixed by suction with a holding pipette, and 5 μg/ml of DNA (EGFP) in 1-2 pl was microinjected using air pressure (Pc -compensation and Pi -injection pressure, with injection time) into one pro-nucleus (single microinjection, SM) or into both pronuclei (double microinjection, DM), as described (Kupriyanov et al, 1998;Chrenek et al, 2005). Swelling of the pronuclei by 10% indicated successful microinjection.…”

“…Individual transgenic embryos carrying either hFVIII gene (detected by PCR analysis as described by Chrenek et al, 2005) or EGFP gene (detected by fluorescence analysis as described by Ikawa et al, 1995 andChrenek et al, 2005) as well as nontransgenic control embryos for analysis were taken from in vivo or in vitro fertilization. The first step involved synchronization of embryo donor females using PMSG and HCG hormones for superovulation purposes.…”