In utero transfer and expression of exogenous genes in sheep

Objective. We have previously reported that directly injecting low-titer re troviral vector supernatant into pre-immune sheep fetuses resulted in the t ransfer and long-term expression of the bacterial Neo(R) gene within the he matopoietic system of these animals for over 5 years. In the present studie s, we investigated whether using a higher titer vector would enable more ef ficient transduction and expression of the transgenes within the hematopoet ic cells in sheep injected in utero, Materials and Methods. Sixteen pre-immune sheep fetuses were injected intra peritoneally with the G1nBgSvNa8.1 helper-free retroviral vector supernatan t encoding the bacterial Neo(R) and LacZ genes (titer: 1 X 10(7) cfu/mL), Results. Over the 2-year time course of these studies, the presence and exp ression of the NeoR and LacZ genes were demonstrated in 12 of the 14 animal s evaluated by several immunological and biochemical methods. Seven of the 12 sheep examined by flow cytometric analysis contained greater than or equ al to 6% transduced peripheral blood lymphocytes, Vector distribution was w idespread without any detectable pathology. Importantly, PCR analyses and b reeding experiments demonstrated that the germ line was not altered. Conclusions. These studies confirmed that direct injection of an engineered retrovirus is a feasible means of safely delivering foreign genes into a d eveloping fetus and thus achieving longterm expression of the transgenes wi thin the recipient's hematopoietic cells, Furthermore, expression of the Ne oR gene from these studies was higher than that reported in our previous st udy in which a lower titer vector was used. (C) 2000 International Society for Experimental Hematology. Published by Elsevier Science Inc.