Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses

Kohli, Manu

doi:10.1093/nar/gnh009

Cited by 146 publications

(146 citation statements)

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“…For isolation of cell lines harboring EGFP‐tagged STIL and Sas‐6, fluorescence signals were too weak to permit sorting by FACS. Thus, STIL‐EGFP and Sas‐6‐EGFP lines were generated using neomycin selection, as described previously (Kohli et al , 2004). In brief, neomycin‐resistant clones were screened by PCR on genomic DNA for correct integration.…”

Section: Methodsmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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Centrioles are essential for the formation of centrosomes and cilia. While numerical and/or structural centrosomes aberrations are implicated in cancer, mutations in centriolar and centrosomal proteins are genetically linked to ciliopathies, microcephaly, and dwarfism. The evolutionarily conserved mechanisms underlying centrosome biogenesis are centered on a set of key proteins, including Plk4, Sas‐6, and STIL, whose exact levels are critical to ensure accurate reproduction of centrioles during cell cycle progression. However, neither the intracellular levels of centrosomal proteins nor their stoichiometry within centrosomes is presently known. Here, we have used two complementary approaches, targeted proteomics and EGFP‐tagging of centrosomal proteins at endogenous loci, to measure protein abundance in cultured human cells and purified centrosomes. Our results provide a first assessment of the absolute and relative amounts of major components of the human centrosome. Specifically, they predict that human centriolar cartwheels comprise up to 16 stacked hubs and 1 molecule of STIL for every dimer of Sas‐6. This type of quantitative information will help guide future studies of the molecular basis of centrosome assembly and function.

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Section: Methodsmentioning

confidence: 99%

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

et al. 2016

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“…Therefore, although it seems likely that downregulation of certain miRNAs may be a feature of tumor cells and de-differentiation, it remains to be seen whether miRNA expression changes are a cause or consequence of neoplasia. One approach to address this issue would be through systematic disruption of miRNA genes in human cancer cells, using somatic gene targeting approaches (Kohli et al, 2004). Alternatively, recent methods may permit knockdown of miRNAs using modified antisense oligonucleotides (Hutvagner et al, 2004;Meister et al, 2004) or siRNAs that target miRNA precursors to evaluate their function.…”

Section: Mirnas and Cancermentioning

confidence: 99%

Implications of micro-RNA profiling for cancer diagnosis

2006

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“…Strategies including positive and negative marker selection and promoter-trap can boost efficiencies considerably, although these approaches present their own technical challenges and are not always successful in achieving high efficiency targeting (4,5). Although advances with adeno-associated viral delivery strategies continue to improve the efficiency of knockouts (6,7), the frequency is still very low and the time required to achieve biallelic gene knockout remains a barrier to its routine adoption. Here, we present a general solution for rapid gene knockout in mammalian cells.…”

mentioning

confidence: 99%

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

Santiago

Chan²,

Liu³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

329

230

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Gene knockout is the most powerful tool for determining gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for gene disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target gene is first cleaved by the ZFNs and then repaired by a natural-but imperfect-DNA repair process, nonhomologous end joining. This often results in the generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic gene disruption at frequencies >1%, thus obviating the need for selection markers. Three new genetically distinct DHFR ؊/؊ cell lines were generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR gene. Importantly, target gene disruption is complete within 2-3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR ؊/؊ cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for gene knockout with application to reverse genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.genetic engineering ͉ zinc-finger proteins T he use of gene knockouts in basic research, functional genomics, and industrial cell line engineering is severely limited by an absence of methods for rapid targeting and disruption of an investigator-specified gene. Early approaches to somatic cell gene disruption used genome-wide nontargeted methods, including ionizing radiation and chemical-induced mutagenesis (1, 2) whereas more recent methods used targeted homologous recombination (HR) (3). However, the Ͼ1,000-fold lower frequency of the targeted HR event relative to random integration in most mammalian cell lines (beyond mouse ES cells) can necessitate screening thousands of clones and take several months to identify a biallelic targeted gene knockout. Strategies including positive and negative marker selection and promoter-trap can boost efficiencies considerably, although these approaches present their own technical challenges and are not always successful in achieving high efficiency targeting (4, 5). Although advances with adeno-associated viral delivery strategies continue to improve the efficiency of knockouts (6, 7), the frequency is still very low and the time required to achieve biallelic gene knockout remains a barrier to its routine adoption. Here, we present a general solution for rapid gene knockout in mammalian cells.The repair of double strand DNA breaks (DSB) in mammalian cells occurs via the distinct mechanisms of homol...

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Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses

Cited by 146 publications

References 20 publications

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Quantitative analysis of human centrosome architecture by targeted proteomics and fluorescence imaging

Implications of micro-RNA profiling for cancer diagnosis

Targeted gene knockout in mammalian cells by using engineered zinc-finger nucleases

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