A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells

Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, PR China.
Genome biology (Impact Factor: 10.81). 02/2007; 8(4):R48. DOI: 10.1186/gb-2007-8-4-r48
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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.
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.
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 virus.

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    • "In addition, it has been demonstrated that human ES (hES) cells are susceptible to coxsackievirus B and produce viable virus particles (Scassa et al., 2011). However, apart from work with retroviral vectors (Wang & Bradley, 2007), less is known about the ability of human viruses to productively infect and replicate in mES cells. Here, we characterized virus infection of mES cells using influenza A virus and herpes simplex virus type 1 (HSV-1). "
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    ABSTRACT: Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence.
    Journal of General Virology 07/2012; 93(Pt 10):2118-30. DOI:10.1099/vir.0.043406-0 · 3.18 Impact Factor
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    • "The libraries used for this screen were generated in Blm-deficient ES cells which facilitates the segregation of homozygous mutants from pools of single allele mutations. This genetic background has supported several successful screens [8], [9], [18]. The mutation libraries used for this screen were generated using the PiggyBac transposon to insert a conventional gene-trap cassette into the ES cell genome [10]. "
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    ABSTRACT: Host gene products required for mediating the action of toxins are potential targets for reversing or controlling their pathogenic impact following exposure. To identify such targets libraries of insertional gene-trap mutations generated with a PiggyBac transposon in Blm-deficient embryonic stem cells were exposed to the plant toxin, ricin. Resistant clones were isolated and genetically characterised and one was found to be a homozygous mutant of the mannosidase 2, alpha 1 (Man2α1) locus with a matching defect in the homologous allele. The causality of the molecular lesion was confirmed by removal of the transposon following expression of PB-transposase. Comparative glycomic and lectin binding analysis of the Man2α1 (-/-) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal β-galactose moieties, potential target receptors for ricin. Furthermore, naïve ES cells treated with inhibitors of the N-linked glycosylation pathway at the mannosidase 2, alpha 1 step exhibited either full or partial resistance to ricin. Therefore, we conclusively identified mannosidase 2, alpha 1 deficiency to be associated with ricin resistance.
    PLoS ONE 08/2011; 6(8):e22993. DOI:10.1371/journal.pone.0022993 · 3.23 Impact Factor
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    • "In principle, mutants of any gene expressed in Blm-deficient ES cells can be isolated from these libraries with an appropriate screening strategy. Previously we have described two screens (Guo et al. 2004; Wang and Bradley 2007) conducted in Blm-deficient ES cells (Luo et al. 2000). These screens used sectored libraries that were composed of 10,000 unique gene-trap mutations constructed with a reversible gene-trap retroviral vector (RGTV-1). "
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    ABSTRACT: Cultured mouse or human embryonic stem (ES) cells provide access to all of the genes required to elaborate the fundamental components and physiological systems of a mammalian cell. Chemical or insertional mutagenesis of Blm-deficient mouse ES cells can be used to generate genome-wide libraries of homozygous mutant ES cells, which are the substrates for conducting phenotype-driven loss-of-function genetic screens. However, the existing insertional mutation libraries are limited by incomplete genomic coverage. In this study, we have explored the use of piggyBac (PB) transposon-mediated mutagenesis to extend the genomic coverage of mutation libraries in Blm-deficient ES cells. A library composed of 14,000 individual gene-trap clones was generated and a recessive genetic screen conducted to identify cells with defects in DNA mismatch repair (MMR) genes. Independent mutations in all known genes of the pathway Msh2, Msh6, Pms2, and Mlh1 were recovered in these screens. The genomic coverage in this library confirms its utility as a new genetic resource for conducting recessive genetic screens in mammalian cells.
    Genome Research 03/2009; 19(4):667-73. DOI:10.1101/gr.085621.108 · 14.63 Impact Factor
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