Galpin/DA/N2 DIBenv 
purified formulated HIV-IT (V). Mice were sacrificed 24 hours and 72 hours post 
injection. The injection site, contralateral muscle, spleen, liver, kidneys, thymus and 
gonads (testes from male, ovaries from female) were removed from each animal at the 
time of sacrifice and immediately frozen. In preliminary studies, we have examined 
injection site muscle samples from 8 animals sacrificed 72 hours post-injection with 
HIV-IT (V) and muscle from a control non-injected animal. HIV-IT (V) DNA was 
detected by PCR amplification of the HIV-1 env gene in the DNA isolated from injection 
sites taken from seven of the mice (Figure 17). DNA was not recovered from the final 
animal in this group. Amplified product was not detected in reactions containing DNA 
isolated from the muscle taken from the negative control (non-injected) mouse. The 
sensitivity of the assay is estimated at 1 copy per 10^ cells from separately performed 
spiking experiments. Individual experiments were controlled by spiking 2.5 and/or 5 
copies per 10^ cell equivalent(s) of DNA. These results show we can detect HIV-1 env 
sequences encoded by HIV-IT (V) at the injection site. 
To determine the time course of the appearance of HIV-1 env DNA and its 
subsequent clearnace, tissue samples from HIV-IT (V) injected mice were obtained at 
various timepoints after to vector administration. In a separate study, four groups of five 
male mice were injected IM one time with either diluent control ( 1 mouse per group) or 
with 0.1 ml (1 x 10 6 cfu) of purified, formulated HIV-IT(V) (4 mice per group). One 
group (4 experimental, 1 control) of mice was sacrificed at each time point post-injection: 
2 hours, 24 hours, 7 days or 21 days. The injection site, contralateral muscle, spleen, 
liver, kidneys, thymus and gonads were removed from each animal at the time of sacrifice 
and immediately flash frozen. The presence of HIV-IT provector was determined using 
PCR amplification of the HIV-1 env gene in DNA isolated from the injection sites taken 
from mice sacrificed at each timepoint. 
The results indicate that transduction of cells at the site of injection is detectable after 
2 hours in four out of four mice tested. In vitro studies of the retroviral’life cycle indicate 
Recombinant DNA Research, Volume 17 
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