402 OOCYTE-MEDIATED GENE TRANSFER: A NOVEL APPROACH TO PRODUCE TRANSGENIC PORCINE EMBRYOS

Abstract

Classical approaches for producing transgenic livestock require labor-intensive, time-consuming, and expensive methods but have low transgenic efficiency and a high mosaicism rate. This study evaluated a simplified method for producing transgenic porcine embryos by microinjecting a DNA construct into unfertilized metaphase oocytes that were subsequently fertilized in vitro. For this, oocytes recovered from abattoir-derived prepubertal porcine ovaries were matured in vitro for 42–44 h and were microinjected with DNA solution (10 ng µL^-1) using a femtojet microinjector (Eppendorf, Hamburg, Germany). The DNA (4.7 kb) was derived from the pEGFP-C1 plasmid (Clontech Laboratories Inc., Palo Alto, CA, USA), which contains the enhanced green fluorescent protein (EGFP) encoding transgene under the control of cytomegalovirus promoter, and linearized with ApaLI restriction enzyme. Injected oocytes were then in vitro-fertilized using fresh epididymal sperm obtained from abattoir-derived porcine testis by standard procedure and cultured in NSCU23 medium supplemented with 0.4% BSA. The efficiency of transgenesis was monitored by visualization of green florescence under UV illumination using a EGFP filter set. Data were analyzed by Student's t-test. Results showed that the cleavage rate of injected oocytes (68.7 ± 0.5%) was similar to that of non-injected control oocytes (67.8 ± 0.4%). However, a high percentage of injected oocytes showed a developmental block at the 2–4 cell stage. The EGFP expression rate at 2–4 cell stage, when expressed as proportion of injected oocyte, was 17.2 ± 0.1%. Interestingly, mosaicism was not observed. The EGFP expression rate increased to 26.7 ± 0.1% when the DNA concentration was increased to 40 ng µL⁻¹. Injecting the DNA solution near the metaphase plate of the oocyte did not improve (P < 0.05) the EGFP expression rate (22.2 ± 0.1%). A high proportion of EGFP-expressing oocytes blocked at the 4–8 cell stage and did not progress to blastocyst, possibly due to random integration of the transgene in developmentally important gene loci. Our results thus suggest oocyte-mediated gene transfer as a promising tool for producing transgenic livestock. However, further research is required to improve its efficiency.

This work was supported by the Research Project on the Production of Bio-Organs (No. 200503030201), Ministry of Agriculture and Forestry, Republic of Korea.