Abstract:
The aim of this research was to develop an efficient screening technique to detect transgenic ovine embryos using neomycin resistance (NeoR) and enhanced green fluorescence protein (EGFP) genes as genetic markers. A 0.8-kb fragment of the ovine β-lactoglobulin promoter sequence (BLG) and 1.8 kb of the human augmenter of liver regeneration (ALR) genomic sequence were derived by PCR amplification. These 2 fragments were inserted into the MCS of pGEM-7zf(+) plasmid; this vector was named p7Z-BA. The coding sequence of NeoR was derived by PCR amplification from the plasmid pIRES2-EGFP and was assembled into the MCS of the pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a NeoR gene coding sequence and an EGFP coding sequence linked by an internal ribosome entry site (IRES) sequence downstream of the CMV promoter. The vector pNIE was excised as an NsiI-SspI fragment and inserted into the vector p7Z-BA. In the end, we had a vector named pNEA, which contained the NeoR gene and the EGFP gene regulated by a CMV promoter for expression in a non-tissue specific mode, and the human ALR gene regulated by the BLG promoter for expression specifically in mammary gland. Sheep fetal fibroblast (SFFB) cells were isolated by attachment of tissue pieces from the ear skin of a 1- to 2-month ovine fetus. Karyotypes of the cells at the third passage and after 15 passages were analyzed. The cells proliferated well and more than 72% of the cells maintained a diploid karyotype after 15 passages. Therefore, the SFFB cells are amenable for transgenic cloning manipulations. For transfection, third-passage SFFB cells at 70% confluency were transfected in a 100-mm dish with pNEA (0.5, 1.0, 2.0, 3.0, 5.0, and 7.0 µg) using Lipofectamine 2000 (2, 4, 6, 8, 10, and 12 µL; Invitrogen, Carlsbad, CA). Cells were checked 24 to 48 h after transfection under fluorescence microscopy for GFP expression, and G418 selection (800 µg mL-1) was applied at that time. After 2 weeks, selected colonies were counted and propagated in culture medium containing 300 µg mL-1 G418 for 2 to 3 passages and cryopreserved. A small portion of the cells was analyzed by PCR for gene integration. Bright green fluorescence could be detected 24 to 48 h after transfection. More colonies were selected when transfection parameters were 2 µg of DNA and 10 µL of Lipofectamine. The results of PCR detection showed that the foreign gene was integrated into the genome. A total of 612 oocytes were aspirated from 2- to 5-mm follicles of ovine ovaries collected from an abattoir; 78% of them were matured after 18 h in culture. Four hundred forty-three oocytes were enucleated, and 332 enucleated oocytes were treated for electrofusion with green fluorescence cells. Of these, 180 (54.2%) couplets were fused. A total of 172 reconstructed embryos were stimulated and cultured in vitro, 31 (18%) of which developed to the blastocyst stage, and 19 blastocysts expressed GFP. In conclusion, we established an effective method to select transgenic embryos formed by nuclear transfer using transfected donor cells.