P-796 A novel method of somatic cell nuclear transfer to generate artificial oocytes

Abstract

Abstract Study question Can we identify an alternative method to Sendai-Virus fusion for somatic cell nuclear transfer (SCNT) aiming at generating artificial oocyte? Summary answer The utilization lipofectamine proved itself as a valid alternative method to virus-mediated SCNT, allowing successful membrane fusion and supporting proper embryo development. What is known already SCNT involves transferring a diploid cell’s nucleus into an enucleated oocyte. Early attempts of SCNT involves direct injection and electrofusion but both approaches can affect oolemma integrity. To avoid mechanical damage to oolemma, fusogenic agents have been employed for SCNT including polyethylene glycol (PEG) and more efficient Hemagglutinating Virus of Japan Envelope (HVJ-E). However, despite its high efficiency, HVJ-E is difficult to be used in human because it is a virus derivative, and a recent shortage of this compound made it not readily available. On the other hand, cationic phospholipid is fusogenic and is widely utilized in transfection experiments. Study design, size, duration In the past 4 months, SCNT was carried out through PEG-mediated, HVJ-E-mediated, or novel lipofectamine-mediated fusion using cumulus cells as nuclear donor. Post-SCNT oocytes were inseminated and cultured up to the blastocyst stage. Embryo development and morphokinetics were assessed and compared among the three SCNT experimental cohorts. In some cases, blastocysts were transferred into pseudo-pregnant mice, post-natal development were monitored. Fertilization, embryo development, and offspring wellbeing were monitored. Participants/materials, setting, methods Oocytes from B6D2F1 mice were enucleated by micromanipulation. The ooplasts were allocated to 3 cohorts: 1) exposure to 45% PEG, 2) subzonal transfer of donor cell with HVJ-E, or 3) subzonal transfer the donor cell exposed to 1.75% lipofectamine. Reconstructed oocytes were monitored for fusion 30 minutes after exposure to fusogens and inseminated by spermatozoa from B6-EGFP mice to confirm paternal genome provenance. Fertilization and embryo development were monitored in a time-lapse system. Main results and the role of chance A total of 165 oocytes were enucleated and 160 (97.0%) survived and allocated to PEG-fusion (n = 50), Sendai-virus-fusion (n = 41) and lipofecamine-fusion (n = 80) cohorts. Fusion rate was 56.0% (28/50) for the PEG-fusion cohort, while Sendai-virus and lipofectamine-fusion cohorts yielded higher fusion rate at 92.7%(38/41) and 86.3%(69/80), respectively (P<0.0001). Control oocytes yielded 96.7% (145/150) fertilization rate and developed to 2-cell stage at 93.8% (136/145), and yielded an eventual blastocyst rate at 80.7% (117/145). Despite a lower oocyte reconstruction rate, PEG-fusion cohort fertilized at 89.3% (25/28), but cleavage rate to 2-cell stage was lower at 48.0% (12/25, P<0.0001) and did not yield any blastocyst. The HVJ-E cohort yielded a fertilization rate of 94.7% (36/38) and cleavage rate at 97.2% (35/36) similar to control. The blastocyst rate was observed at 22.9% (8/35, P<0.0001) when comparing to control. Lipofectamine-fusion cohort yielded 91.3% (63/69) fertilization rate and subsequent cleavage rate at 95.2% (60/63). Albeit at a lower blastocyst development rate comparing to control, the lipofectamine cohort yielded similar full-preimplantation rate at 19.0% (12/63) when comparing to HVJ-E cohort. All blastocysts (n = 20) were transferred into 2 pseudo-pregnant mice and 3 healthy female offspring were yielded to date. Limitations, reasons for caution HVJ-E mediated membrane fusion has been well established with proven safety record; however, its recent shortage has led to a temporary cessation of its utilization. Our lipofectamine-mediated cell fusion technique yielded satisfactory fusion, fertilization, blastocyst formation and some live offspring, however the safety and efficacy still remain to be validated. Wider implications of the findings This novel lipofectamine-mediated cell fusion has broader implications for advancing cloning technologies. Lipofection is a non-viral approach, overcomes shortage of HVJ-E and therefore propose itself as a more appealing cell fusion method for human SCNT. Trial registration number N/A