376 VECTOR-MEDIATED GENE TRANSFER INTO RHESUS MACAQUE EMBRYOS

Abstract

A series of experiments was performed to assess the suitability of various viral vectors for transformation of rhesus macaque (Macaca mulatta) embryos. Viral vectors included the adenovirus-associated virus (AAV) containing a CMV-EGFP transgene and a lentivirus (FUGW) carrying the green fluorescent protein (GFP) gene from either the jellyfish or a humanized version from renilla linked to an ubiquitin promoter. Embryos were generated by in vitro fertilization of oocytes retrieved by laparoscopy from superovulated females. Resulting zygotes were injected under the zona pellucida (ZP) with varying viral concentrations. Embryos were subsequently cultured for 5 days and thereafter analyzed by epifluorescence microscopy. A total of 62 zygotes were injected with one of three vectors AAV2 (25), AAV2.1 (15), or AAV2.7 (22). Of these 22, 9 and 16, respectively, reached the blastocyst and morula stage. There was no difference in the percentage of embryos expressing GFP between vectors (24, 53.2, and 36.4%, respectively). However, all of the positive embryos proved to be mosaics. In a second set of experiments, FUGW was injected under the ZP of 155 embryos. Of these, 76 received virus carrying renilla GFP, while the remaining 79 were injected with virus carrying the jellyfish GFP. Following injection with renilla, 52 reached the blastocyst/morula stage on Day 7, while 43 containing jellyfish GFP proceeded to this stage. Expression of jellyfish GFP could be seen in 65% of the embryos of which 35.6% were mosaics, whereas renilla GFP was found in only 15.8% of the embryos, although none of these were mosaic. To determine whether the mosaic expression was caused by transgene silencing, three mosaic embryos were dissociated on Day 3 and 10; 10 and 8 blastomeres, respectively, were obtained. Analysis by PCR showed all but one blastomere carrying the vector. Similarly, the presence of the vector was identified by PCR in 17 of 19 non-expressing embryos injected with renilla. These results show that AAV and lentivirus can transform rhesus embryos, which can subsequently continue in development. However, identification of positive embryos by epifluorescence alone may not be sufficient.

Funding was provided by NIH/NCRR grant RR000164-13.