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A reversible gene trap collection empowers haploid genetics in human cells.

ABSTRACT: Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expressed genome, is DNA-barcoded and allows systematic screens for a wide variety of cellular phenotypes. We examined cellular responses to TNF-?, TGF-?, IFN-? and TNF-related apoptosis-inducing ligand (TRAIL), to illustrate the value of this unique collection of isogenic human cell lines.

SUBMITTER: Burckstummer T

PROVIDER: S-EPMC6342250 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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A reversible gene trap collection empowers haploid genetics in human cells.

Bürckstümmer Tilmann T Banning Carina C Hainzl Philipp P Schobesberger Richard R Kerzendorfer Claudia C Pauler Florian M FM Chen Doris D Them Nicole N Schischlik Fiorella F Rebsamen Manuele M Smida Michal M Fece de la Cruz Ferran F Lapao Ana A Liszt Melissa M Eizinger Benjamin B Guenzl Philipp M PM Blomen Vincent A VA Konopka Tomasz T Gapp Bianca B Parapatics Katja K Maier Barbara B Stöckl Johannes J Fischl Wolfgang W Salic Sejla S Taba Casari M Rita MR Knapp Sylvia S Bennett Keiryn L KL Bock Christoph C Colinge Jacques J Kralovics Robert R Ammerer Gustav G Casari Georg G Brummelkamp Thijn R TR Superti-Furga Giulio G Nijman Sebastian M B SM

Nature methods 20130825 10

Knockout collections are invaluable tools for studying model organisms such as yeast. However, there are no large-scale knockout collections of human cells. Using gene-trap mutagenesis in near-haploid human cells, we established a platform to generate and isolate individual 'gene-trapped cells' and used it to prepare a collection of human cell lines carrying single gene-trap insertions. In most cases, the insertion can be reversed. This growing library covers 3,396 genes, one-third of the expres ...[more]

PMID: 24161985

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Project description:BackgroundConditional gene knockout (cKO) mediated by the Cre/LoxP system is indispensable for exploring gene functions in mice. However, a major limitation of this method is that gene KO is not reversible. A number of methods have been developed to overcome this, but each method has its own limitations.ResultsWe describe a simple method we have named LOFT [LoxP-flippase (FLP) recognition target (FRT) Trap], which is capable of reversible cKO and free of the limitations associated with existing techniques. This method involves two alleles of a target gene: a standard floxed allele, and a multi-functional allele bearing an FRT-flanked gene-trap cassette, which inactivates the target gene while reporting its expression with green fluorescent protein (GFP); the trapped allele is thus a null and GFP reporter by default, but is convertible into a wild-type allele. The floxed and trapped alleles can typically be generated using a single construct bearing a gene-trap cassette doubly flanked by LoxP and FRT sites, and can be used independently to achieve conditional and constitutive gene KO, respectively. More importantly, in mice bearing both alleles and also expressing the Cre and FLP recombinases, sequential function of the two enzymes should lead to deletion of the target gene, followed by restoration of its expression, thus achieving reversible cKO. LOFT should be generally applicable to mouse genes, including the growing numbers of genes already floxed; in the latter case, only the trapped alleles need to be generated to confer reversibility to the pre-existing cKO models. LOFT has other applications, including the creation and reversal of hypomorphic mutations. In this study we proved the principle of LOFT in the context of T-cell development, at a hypomorphic allele of Baf57/Smarce1 encoding a subunit of the chromatin-remodeling Brg/Brahma-associated factor (BAF) complex. Interestingly, the FLP used in the current work caused efficient reversal in peripheral T cells but not thymocytes, which is advantageous for studying developmental epigenetic programming of T-cell functions, a fundamental issue in immunology.ConclusionsLOFT combines well-established basic genetic methods into a simple and reliable method for reversible gene targeting, with the flexibility of achieving traditional constitutive and conditional KO.

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A reversible gene trap collection empowers haploid genetics in human cells.

Publications

A reversible gene trap collection empowers haploid genetics in human cells.

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