Biology Reference
In-Depth Information
the RD114 ASCT2 receptor (Rasko
., 1999b), FeLV-C
FLVCR1 receptor from human and domestic cat cDNA libraries (Quigley
et al
., 1999; Tailor
et al
et al
.,
2000; Tailor
., 1999c), and two closely related human proteins, PHuR-A1
and PHuR-A2, that function as receptors for PERV-A (Ericsson
et al
., 2003).
Briefly, when a cell line that is not susceptible to a particular pseudotype
retrovirus vector harboring an envelope for which the receptor is not known, a
cDNA library from a cell line highly susceptible to transduction, was constructed
by cloning the cDNA into a retroviral expression vector. Afterward, the cDNA
retroviral expression library was transduced into nonsusceptible cells by infection
at a relatively low multiplicity of infection so that the majority of infectants
would contain single-copy provirus inserts. The library-containing cells were
then screened for susceptibility to pseudotype vector transduction through selec-
tion of drug-resistant cells after exposure to the vector carrying a resistance gene.
Of drug-resistant clones obtained from the primary screen and using PCR primers
specific for vector sequences, cDNA products from clones with conferred suscep-
tibility were identified after nested PCR was performed on DNA extracted from
reinfectable clones.
However, for many viruses, initial attempts using a retroviral cDNA
library were unsuccessful due to an inherent background of nonspecific infection
with pseudoparticles. In fact, no cell line was completely nonpermissive to even
“no envelope” pseudoparticles bearing no viral envelope proteins, indicating the
existence of nonspecific uptake mechanisms. In the screens, this resulted in a
high background of drug-resistant colonies, independent of glycoprotein-
mediated cell entry. Thus, unless the entry factor cDNA was highly represented
in the library, a single round of transduction/challenge would not suffice. To deal
with the high background observed in screens, methods that would allow multi-
ple rounds of selection and enrichment have been developed. Recently, the use
of a cyclic packaging rescue (CPR) system using non-self-inactivating vectors
has been shown to increase the efficiency of receptor cloning with powerful
iterative screening methods (Evans
et al
., 2009). Most retroviral
vectors commonly used for gene-delivery applications are self-inactivating vec-
tors that contain deletions in the long terminal repeat (LTR) elements. No
packaging competent retroviral RNA transcripts are generated from such
integrated proviruses; instead, transgene expression is driven by an internal
nonretroviral promoter. In contrast, if the cDNA library is constructed in a
provirus that retains the complete LTR elements, the retroviral promoter is
active in transduced cells and a full-length viral RNA is expressed. Expression
of the packaging components, gag-pol and vesicular stomatitis virus VSV-G
envelope (that will efficiently infect recessive cell lines), in these cells allows
packaging of the RNA into pseudoparticles capable of transducing naive cells.
This approach, termed CPR (Bhattacharya
et al
., 2007; Ploss
et al
et al
., 2002; Koh
et al
., 2002), allows
retrieval of
the library after
selection has been performed,
followed by
