400 PRODUCTION OF TRANSGENIC CLONED DOMESTIC CAT EMBRYOS BY LENTIVECTOR-MEDIATING TRANSGENESIS

Abstract

The domestic cat is a useful biomedical model because several cat diseases are analogous to inherited human disorders. Transgenic embryos have been produced by microinjecting lentivirus vectors (LV) carrying specific genes into the perivitelline space of mature oocytes or zygotes of different mammalian species (Hofmann et al. 2003 EMBO Rep. 4, 1054). One drawback of this approach is that integration of the transgene may not occur in each blastomere, and that mosaic embryos are formed (Kubish et al. 2006 Reprod. Fertil. Dev. 18, 295). Donor nuclei derived from cells stably transduced with a LV may provide a more effective strategy for producing transgenic animals via nuclear transfer (NT). The purpose of the present study was to determine the uselfulness of LV to deliver transgene into cat fetal fibroblasts (CFF) and to produce transgenic domestic cat cloned embryos expressing enhanced green fluorescence protein (eGFP). CFF were transduced with LV carrying the eGFP transgene. The LV-construct contained either the human cytomegalovirus (CMV) or the human translation elongation factor 1 alpha (hEF1alpha) promoter to achieve ubiquitous expression of the eGFP transgene. CFF at passage 1–2 were transduced with either LV-CMV or LV-hEF1alpha for 24 h. Cells expressing eGFP were observed at 24 and 48 h after co-incubation with the LV. Stable transgene expression in transduced CFF was observed and only those CFF that fluoresced green by epifluorescence microscopy with a fluorescein isothiocyanate (FITC) filter were selected for NT. Cleavage rate and embryo development to blastocyst stage (Day 8), respectively, of embryos reconstructed with transduced CFF from LV-CMV (79%; 12%) and LV-hEF1alpha (77%; 29%) were not different from those of cloned embryos reconstructed with non-transduced CFF cells (81%; 19%). None of the LV-CMV-derived cloned embryos expressed detectable levels of eGFP, whereas 18% of the LV-hEF1alpha-cloned embryos expressed detectable eGFP. Fluorescence in cloned embryos reconstructed with LV-hEFP1alpha promoter was not observed during the first 24–36 h, but from Day 2, three embryos (9%) at the 2-cell stage started to express eGFP. Two embryos fluoresced brightly and retained fluorescence through development to the morula stage at Day 7. The third embryo had faint levels of fluorescence until Day 5. On day 5, three other embryos (9%) showed faint fluorescence that disappeared by Day 7. Blastocysts at Day 8 derived from either construct did not exhibit green fluorescence. To analyze lentiviral integration and the number of proviral integrants, real-time PCR quantification was performed on genomic DNA of single blastocysts. The number of provirus copies present in the genome of LV-CMV-(n = 4) and LV-hEF1alpha-(n = 6) derived cloned blastocysts ranged from 5 to 9 and 3 to 9 copies, respectively, whereas cloned blastocysts (n = 2) using non-transduced CFF were negative. In summary, we have established that transgenic domestic cat cloned embryos can be produced. All cloned blastocysts derived from either LV construct carried the provirus. However, eGFP expression was not observed in the blastocysts, possibly due to transgene silencing.