title: A recessive genetic screen for host factors required for retroviral infection in a library of insertionally mutated Blm-deficient embryonic stem cells.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, PR China. wxw@sanger.ac.uk
authors.forename: Wei
authors.initials: W
authors.lastname: Wang
authors.forename: Allan
authors.initials: A
authors.lastname: Bradley
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics*; D000818:Animals; D015220:Calcium Channels / Q000235:genetics* / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000821:virology*; D005808:Genes, Recessive; D005819:Genetic Markers; D005822:Genetic Vectors; D007113:Immunity, Innate / Q000235:genetics; D019426:Integrases; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000254:growth & development*; D051379:Mice; D016254:Mutagenesis, Insertional; D053484:RecQ Helicases; D011991:Receptors, Virus / Q000235:genetics*; D050916:TRPV Cation Channels / Q000235:genetics* / Q000502:physiology
chemical_list: D015220:Calcium Channels; D005819:Genetic Markers; D011991:Receptors, Virus; D050916:TRPV Cation Channels; C446923:Trpv6 protein, mouse; C045073:Cre recombinase; D019426:Integrases; D000251:Adenosine Triphosphatases; C097151:Bloom syndrome protein; D004265:DNA Helicases; D053484:RecQ Helicases