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.