333 OCT-4 EXPRESSION ANALYSIS IN F₀ AND F₁ PORCINE OG2 TRANSGENICS

Abstract

OG2 transgenic pigs provide a new large animal model in which to study Oct-4 expression and the derivation, migration and maintenance of pluripotent cells. They may also prove to be a valuable tool for the development of cell-based therapies. The OG2 transgene consists of the genomic sequence of the murine Oct-4 gene with the enhanced green fluorescence protein (EGFP) reporter gene inserted between the promoter and the coding sequences. As previously reported, 11 OG2 founder animals were produced (7 male and 4 female). Two of the OG2-F₀ transgenic boars were mated with 3 wild-type sows and with 2 OG2-F₀ transgenic sows. The pregnancy of 1 wild-type sow was terminated at Day 5 after fertilization, and approximately 60% (14/23) of the flushed blastocysts expressed EGFP, demonstrating germ line transmission. The remaining 2 wild-type sows delivered 21 piglets, of which 11 were transgenic. The 2 OG2-F₀ sows delivered 9 piglets, all of which were transgenic. Transgenesis and tissue-specific expression of the transgene were determined by Southern blotting, Northern blotting, and real-time PCR analysis. Germ cell-specific expression of the OG2 construct was confirmed in both F₀ and F₁ transgenics by fluorescence microscopy. Testis isolated from male transgenic piglets exhibited weak EGFP fluorescence in some cells within the seminiferous tubules, whereas testis tissue from adult transgenic boars gave strong EGFP expression in pre-spermatogonial cells. In contrast, fluorescence-activated cell sorting (FACS) analysis and fluorescence microscopy of ejaculated spermatozoa from 3 mature OG2-F₀ boars displayed no EGFP fluorescence, as expected. Northern blot analysis of EGFP mRNA revealed stronger EGFP expression in the testis of adult transgenic pigs than in the testis from transgenic piglets. No EGFP mRNA was detected in other organs or in control testis isolated from wild-type piglets. Real-time PCR and Northern blot analysis showed that the time course and signal intensity of EGFP expression in OG2 testis paralleled expression of the endogenous Oct-4 gene in both transgenic and in wild-type testis, confirming that there is indeed stronger expression of Oct-4 in the adult testis than in testis from younger animals. We conclude that the OG2 founders exhibit germline transmission and that the offspring express EGFP in a pattern that faithfully mimics expression of the endogenous Oct-4 gene, thus providing a marker for pluripotent cells. We are currently using FACS to isolate EGFP-positive germ cells (pre-spermatogonial stem cells) from the testis of OG2 boars for further characterisation.