Optimization of Transgenic Rabbit Embryo Production Using Pronuclear and Intracytoplasmic Gene Injection Techniques

Abstract

This study compared three microinjection-based gene transfer methods intracytoplasmic sperm injection mediated transgenesis (ICSI-t), intracytoplasmic gene injection (ISI), and pronuclear injection (PNI) using a hyperactive piggyBac plasmid encoding green fluorescent protein (GFP). Female New Zealand White rabbits received 75 IU HMG for three consecutive days followed by 100 IU hCG to induce superovulation. Oocytes and zygotes were collected 15–16 hours later. Two DNA concentrations (10 and 20 ng/µl final) were tested for each method. Embryos cultured in TCM-199 with 15 mg/ml BSA at 38.5°C and 5% CO₂ were assessed at 24, 48, 72, 96, and 120 hours for cleavage, blastocyst formation, and GFP expression. The PNI 10 ng/µl group produced the highest blastocyst rate (42.1%, 16/38) with 37.5% (6/16) of those blastocysts GFP-positive. The PNI 20 ng/µl group gave fewer blastocysts (12.9%, 4/31) but a higher proportion expressed GFP (75.0%, 3/4). ICSI-t performed poorly, yielding blastocysts only at the lower concentration (11.1%, 1/9) with no GFP expression. ISI produced intermediate blastocyst rates (10 ng/µl: 11.3%, 6/53; 20 ng/µl: 10.3%, 4/39) but GFP expression in blastocysts only at the lower concentration (66.6%, 4/6). Higher DNA concentration consistently reduced embryo viability across all methods. Pronuclear injection at 10 ng/µl proved the most effective combination for generating transgenic rabbit embryos in vitro.