Genome Biology 2007, 8:R48
2007Wang and Bradley Volume 8, Issue 4, Article R48
A recessive genetic screen for host factors required for retroviral
infection in a library of insertionally mutated Blm-deficient
embryonic stem cells
Wei Wang*† and Allan Bradley†
Addresses: *Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871, PR China. †The Wellcome
Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.
Correspondence: Allan Bradley. Email: email@example.com
© 2007 Wang and Bradley; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Retroviral infection of embryonic stem cells<p>A recessive genetic screen of an insertionally mutated Blm-/- ES cell library identifies host factors required for retroviral infection, and confirms that mCat-1 is the ecotropic murine leukaemia virus receptor in ES cells.</p>
Background: Host factors required for retroviral infection are potential targets for the
modulation of diseases caused by retroviruses. During the retroviral life cycle, numerous cellular
factors interact with the virus and play an essential role in infection. Cultured embryonic stem (ES)
cells are susceptible to retroviral infection, therefore providing access to all of the genes required
for this process to take place. In order to identify the host factors involved in retroviral infection,
we designed and implemented a scheme for identifying ES cells that are resistant to retroviral
infection and subsequent cloning of the mutated gene.
Results: A library of mutant ES cells was established by genome-wide insertional mutagenesis in
Blm-deficient ES cells, and a screen was performed by superinfection of the library at high
multiplicity with a recombinant retrovirus carrying a positive and negative selection cassette.
Stringent negative selection was then used to exclude the infected ES cells. We successfully
recovered five independent clones of ES cells that are resistant to retroviral infection. Analysis of
the mutations in these clones revealed four different homozygous and one compound
heterozygous mutation in the mCat-1 locus, which confirms that mCat-1 is the ecotropic murine
leukemia virus receptor in ES cells.
Conclusion: We have demonstrated the feasibility and reliability of this recessive genetic
approach to identifying critical genes required for retroviral infection in ES cells; the approach
provides a unique opportunity to recover other cellular factors required for retroviral infection.
The resulting insertionally mutated Blm-deficient ES cell library might also provide access to
essential host cell components that are required for infection and replication for other types of
One characteristic of all viruses is dependency for replication
on components synthesized by host cells. All types of virus are
able to subvert the machinery in the host cell for replication
of the viral genome and expression of viral gene products .
Retroviral replication has a unique aspect, namely conversion
Published: 3 April 2007
Genome Biology 2007, 8:R48 (doi:10.1186/gb-2007-8-4-r48)
Received: 15 November 2006
Revised: 19 February 2007
Accepted: 3 April 2007
The electronic version of this article is the complete one and can be
found online at http://genomebiology.com/2007/8/4/R48
R48.2 Genome Biology 2007, Volume 8, Issue 4, Article R48 Wang and Bradley http://genomebiology.com/2007/8/4/R48
Genome Biology 2007, 8:R48
of genomic viral RNA into cellular DNA, which has been
exploited for the development of antiretroviral drugs. Inte-
gration of a retrovirus into the host genome concludes the
early stage of the life cycle, after which the virus can begin to
Study of the host factors that are involved in the retroviral life
cycle is important if we are to gain a detailed understanding
of the interaction between virus and host cell components.
Essential host cell components are potential targets for anti-
viral therapies that could be developed. Many viruses have
small genomes, and so the repertoire of components that can
be exploited as pharmaceutical targets is very limited. More-
over, because of their rapid replication, variants in the viral
genome that overcome the effect of inhibitors will be rapidly
selected, diminishing the effectiveness of antiviral agents.
The main drugs currently used to treat HIV infection are
inhibitors of two viral proteins, namely the reverse tran-
scriptase and the protease (encoded by the viral pol and gag
genes, respectively). Also, inhibitors of the HIV-1 entry and
fusion steps have been used as a third drug class in recent
years . Thus, therapeutic molecules targeting retroviral
host factors would be a potential new route to modulation of
diseases caused by retroviruses. Evidence of the importance
of host factors is provided by individuals who harbor
homozygous mutations in the gene encoding CC chemokine
receptor (CCR)5, who are extremely resistant to HIV infec-
tion. As a result of these observations, human antibodies to
CCR5 and small-molecule CCR5 antagonists are being inves-
tigated as potential HIV therapies [1,4].
Retroviral vectors are widely used as genetic vehicles or as
mutagens in embryonic stem (ES) cells. Comparatively few
studies have described the molecular components that are
essential for the interaction between retroviruses and ES
cells. In previous studies, several host genes required for viral
infection were identified by screening a gene trap library con-
structed in somatic cells . Here we describe a genetic
screen designed to identify host factors in ES cells that are
required for the early phase of the retroviral life cycle. This
recessive screen was conducted in a library of insertionally
mutated Blm-deficient ES cells. The random insertional
mutations in this library were generated using a recombinant
retroviral gene-trap vector, integration into genes of which
predominantly produces a loss of function mutation; the inte-
grated proviral DNA provides a sequence tag for identifying
the mutation . In principle the genome-wide gene-trap
mutations in this library should provide access to mutations
in the subset of genes expressed in ES cells . The Blm
(which encodes Bloom's syndrome protein)-deficient genetic
background of these ES cells is the second important feature
of this mutation library. Recessive genetic screens in a diploid
mammalian genome require an approach to generate cells
with homozygous mutations, which increases the complexity
of most genetic screens because of the low rate of loss of het-
erozygosity (LOH) of single allelic mutations in wild-type ES
cells. However, Blm-deficient ES cells have a 20-fold increase
in the rate of LOH , which offers a major advantage in
recessive screens. Indeed, two reports have described suc-
cessful use of Blm-deficient ES cells to identify recessive
mutations in genes required for DNA mismatch repair  and
the glycosylphosphatidylinasitol-anchor biosynthesis path-
For the screen described here, we have confirmed the utility
of this system in generating genome-wide homozygous muta-
tions to facilitate recessive genetic screens in vitro by identi-
fying mCat-1 as a critical gene in ES cells that is required for
retroviral infection. This screen was conducted by superinfec-
tion with a retroviral vector carrying the puro-Δtk (puromy-
cin-Δ-thymidine kinase) gene, a positive/negative selectable
marker . Clones surviving negative selection were shown
to be resistant to retroviral infection, and in every case the
mCat-1 gene was mutated. This success demonstrates the fea-
sibility of conducting genome-wide negative selection screens
for genes that confer resistance to infection.
Screening strategy for infection resistant mutants
The overall strategy for the screen is illustrated in Figure 1.
The principle behind the screen is selection against retrovi-
rally infected ES cells. The retrovirus used in the screen car-
ried the puro-Δtk positive/negative selection marker .
Infected cells expressing the puro-Δtk fusion gene are sensi-
tive to 1-(-2-deoxy-2-fluoro-1-β-D-arabino-furanosyl)-5-
iodouracil (FIAU) negative selection; thus, mutant ES cells
that cannot be infected by the virus will survive this negative
selection. This screen is therefore strongly dependent on the
ability to infect all cells in the culture with a retrovirus that
reliably expresses a negative selection cassette. A very high
infection efficiency must be achieved so that every single
infectable cell in the culture has at least one infection event.
In practice, the need to infect every cell in a culture of 109 cells
requires superinfection, in which every cell has between 10
and 20 independent viral insertions. Superinfection can be
achieved in a variety of ways, but in the screen described here
it was accomplished by co-cultivation of the gene-trap ES cell
library with the viral producer cells.
Superinfection with a puro-Δtk retroviral vector
To generate a viral producer cell line with a high titre, six dif-
ferent murine leukemia virus (MuLV) backbones were tested.
The puro-Δtk cassette was cloned into each backbone and the
vectors were tested for their efficiency in producing recom-
binant virus by transient transfection into phoenix packaging
cells . Viral titers were assessed using wild type ES cells,
and the WWF6 (Wang Wei female 6) vector had the highest
titer (Figure 2) because of several point mutations and a dele-
tion in its long terminal repeat that prevents transcriptional
suppression in ES cells . This recombinant vector was
used to generate the stable viral producer cell line B4-5 in
Genome Biology 2007, Volume 8, Issue 4, Article R48 Wang and Bradley R48.11
Genome Biology 2007, 8:R48
base PstI-digested fragment from pYTC37 , which is the
plasmid that contains the puro-Δtk cassette in pPGKbpA.
Reverse transcription polymerase chain reaction
Total RNA was extracted from the five mutant clones and
NGG5.3 cells as a negative control, and 5 μg total RNA was
used to generate the first strand cDNA. β-Actin (Actb) was
used as a positive control for RT-PCR. The PCR product of the
mutant mCat-1 transcript from exon 4 to 7 from clone V3 was
TA cloned for sequencing.
Five million ES cells were electroporated with 10 μg of the Cre
expression plasmid pCAG-Cre , plated at low density
(2,000 cells/90 mm plate), and grown without selection for 9
days. ES cell colonies were picked into 96-well plates. Clones
that had excised both copies of the gene-trap cassette were
identified as G418-sensitive clones by sib-selection and con-
firmed by genomic PCR, using primers that amplify a 578
base pair fragment in the βgeo cassette. Reinfection was per-
formed as described above to assess phenotypic reversal.
We would like to thank Ge Guo for discussion at an early phase in the
design of this screen; Zikai Xiong for great help with experimental instruc-
tion; Frances Law and Alistair Beasley for help with tissue culture; and
Antony Rodriguez, Shaun Cowley, and Haydn Prosser for their comments
on this manuscript. This work was supported by the Wellcome Trust &
Sanger Institute grant number 79643.
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