Galpin/DA/N2 IIIBenv 
exist between pSCVIO and pCMVenv am Dra (the plasmid used to express 
amphotropic envelope in the PCL), but these plasmids are derived from two different 
murine leukemia viruses, MoMLV and MLV 4070A, respectively, which differ in their 
nucleotide sequences. This design makes the probability of generating RCR low. 
(d) Testing for Replication-Competent Retroviruses 
A number of novel reagents and procedures have been used to detect RCR in 
conjunction with a commercially available validated extended S + L _ test. The tests 
used are summarized below. 
Test Measurement 
Extended S + L _ Focus formation 
Mus dunni marker rescue (MdH) Hygromycin resistance (hygro r ) 
Mus dunni marker rescue (MdC) B-gal gene expression 
Mus dunni cocultivation with PCL 
Marker rescue or S+L - on the cell 
supernatant. 
The marker rescue assays test vector preparations using a Mus dunni cell line that 
has been transduced with a retroviral vector encoding either hygro r (MdH) or 
beta-galactosidase (MdC). This cell line was chosen as it is readily infected by ampho- 
, eco- (except MoMLV), xeno- and poly-tropic retroviruses and is very permissive for 
propagation of RCR. When MdH cells are infected with a RCR, the virus "rescues", 
by transcomplementation, the hygro r retrovirus which can then transduce a target cell. 
If the target cells are selected with hygromycin, formation of resistant colonies 
indicates the presence of RCR. The MdC assay is similar in principle, however, a 
8-gal containing vector is rescued and assayed on target cells using X-gal staining. All 
HIV-IT (V) preparations tested from the DA/N2 IIIBenv. 2C3 PCL have been negative 
using these assays. In our experience, these assays are roughly comparable in 
sensitivity. The extended S+L' assay which is commercially available, is at least as 
sensitive as other assays. 
Recombinant DNA Research, Volume 17 
[875] 
