7) Preservative-free Protamine sulphate (single use of i.v. 
grade material ) , Elkins-Sinn Inc. NDC# 0641-2554-41, at 
10,000 ug/ml for I.V. use) will be added at 4 ug/ml. 
Flasks will be incubated at 37°C in 5% CO 2 in 80% 
humidity for 6 hours. The flasks will be gently mixed 
every 2 hours. 
8) Following incubation, the cells will be pelleted by 
spinning at 1200 x g for 5 minutes, washed three times, 
and then counted. 
9) After removal of an aliquot of cells for methylcellulose 
cultures (see step #10 below) , the remaining cells will 
be frozen in a programmed rate freezer using standard 
procedures and stored in liquid nitrogen in a separate 
and identical manner to that used for the majority of the 
marrow or peripheral blood which is unwashed. 
10) Colony numbers produced in methylcellulose by an aliquot 
of non-transduced (from step #3) and transduced (from 
step #8) mononuclear cells will be measured in the 
presence or absence of 0.8-1. 2 mg/ml G418 (Gibco license 
of Geneticin; Sobering Corp.). 
11) If there is near or complete failure of growth of the 
transduced marrow (<4 colonies/10^ cells) , patients will 
not be eligible for re-infusion of the transduced portion 
of the marrow. 
12) Colonies will be analyzed for morphology, confirmed if 
necessary by standard staining techniques (reviewed in 
The Hemopoietic Colony Stimulating Factors by Donald 
Metcalf, Elsevier, Amsterdam 1984, and used also in 
previous published studies by Heslop et al, Gottlieb et 
al, and Oblakowski et al referenced previously in 
protocols of Dr. M.K. Brenner) . 
13) Confirmation of transduction in positively identified 
G418 selected and non-selected colonies will be obtained 
by PCR using neo-r gene specific oligonucleotide primers 
which have the following sequence: 
CAAGATGGATTGCACGCAGG and CCCGCTCAGAAGAACTCGTC 
A Southern blot of an electrophoretic gel separation of 
the primer reaction will be generated and tested with a 
probe diagnostic for the viral NEC gene. 
Shown in Figure 1 is the ethidium bromide gel and 
autoradiograph of a gel in which the products of a PCR reaction are 
presented. The template was LNL6 plasmid DNA which contains the 
NEC gene. The first round of amplification utilized the two 
Recombinant DNA Research, Volume 14 
[747] 
