A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting

Konishi, Hiroyuki; Lauring, Josh; Garay, Joseph P.; Karakas, Bedri; Abukhdeir, Abde M.; Gustin, John P.; Konishi, Yuko; Park, Ben Ho

doi:10.1038/nprot.2007.409

Cited by 22 publications

(21 citation statements)

References 26 publications

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“…The strength of the rodent system stems predominately from the ability to make targeted alterations of individual genes using the technology of HR [80]. This technology exists for human somatic cells as well [46],[47],[81]. Overall, at least 77 different genes have been functionally inactivated in a total of 45 different human somatic cell lines {[47] and unpublished data}.…”

Section: Discussionmentioning

confidence: 99%

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

et al. 2010

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The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

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Section: Discussionmentioning

confidence: 99%

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

et al. 2010

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“…Cell lines used have been described (31). Overexpression and shRNA constructs used for transfection and assays were performed as described (37,38).…”

Section: Methodsmentioning

confidence: 99%

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

Mohseni

Cidado

Croessmann

et al. 2014

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

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Tamoxifen is effective for treating estrogen receptor-alpha (ER) positive breast cancers. However, few molecular mediators of tamoxifen resistance have been elucidated. Here we describe a previously unidentified gene, MACROD2 that confers tamoxifen resistance and estrogen independent growth. We found MACROD2 is amplified and overexpressed in metastatic tamoxifen-resistant tumors. Transgene overexpression of MACROD2 in breast cancer cell lines results in tamoxifen resistance, whereas RNAi-mediated gene knock down reverses this phenotype. MACROD2 overexpression also leads to estrogen independent growth in xenograft assays. Mechanistically, MACROD2 increases p300 binding to estrogen response elements in a subset of ER regulated genes. Primary breast cancers and matched metastases demonstrate MACROD2 expression can change with disease evolution, and increased expression and amplification of MACROD2 in primary tumors is associated with worse overall survival. These studies establish MACROD2 as a key mediator of estrogen independent growth and tamoxifen resistance, as well as a potential novel target for diagnostics and therapy.breast cancer | tamoxifen | resistance | MACROD2 | ER positive T he selective estrogen receptor modulator (SERM) tamoxifen is a highly effective drug for the prevention and treatment of estrogen receptor-alpha (ER) positive breast cancers (1). However, resistance to this drug remains a clinically important problem. The molecular mediators of tamoxifen resistance have not been fully elucidated. In part, this is due to the heterogeneous nature of breast cancers, resulting in multiple mechanisms of resistance. For example, past studies have demonstrated that tamoxifen resistance is mediated by differential expression of nuclear hormone receptor coregulators (2, 3), growth factor signaling crosstalk (4-7), regulation of microRNAs (8), cyclin dependent kinases (CKDs) (9), CDK inhibitors (10, 11), and more recently, acquired somatic mutations and alterations in ER (12-17). Further insight into the molecular mediators of tamoxifen and hormone therapy resistance would have great impact on the ability to target genes and pathways that could overcome drug resistance and lead to improved clinical outcomes.In this study we describe a previously unidentified gene, MACROD2, which is amplified and overexpressed in a subset of breast cancers. MACROD2 belongs to a family of genes containing a macro domain, an evolutionarily conserved protein motif (18), whose functional role until recently has been unclear. Studies have demonstrated that MACROD2 deacetylates O-acetyl-ADP ribose, a signaling molecule generated by the deacetylation of acetylated lysine residues in histones and other proteins (19). More recent work demonstrates that MACRO domain containing proteins are involved with mono-ADP ribosylation, and can regulate cell signaling pathways and modify proteins involved with gene transcription (20). Interestingly, MACROD1 (LRP16) has been implicated in modulating ER and androgen receptor (AR) signaling in prio...

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“…The resulting targeting vector was transduced into MCF-10A and hTERT-IMEC, as previously described (46,47). BRCA1 gene-targeted clones were isolated by PCR-based screening and processed for Cre-loxP recombination to remove neomycin-resistance gene cassettes, as previously described (46)(47)(48). Primer sequences for PCR are provided in Table S5.…”

Section: Methodsmentioning

confidence: 99%

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells

Konishi

Mohseni

Tamaki

et al. 2011

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

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Biallelic inactivation of cancer susceptibility gene BRCA1 leads to breast and ovarian carcinogenesis. Paradoxically, BRCA1 deficiency in mice results in early embryonic lethality, and similarly, lack of BRCA1 in human cells is thought to result in cellular lethality in view of BRCA1's essential function. To survive homozygous BRCA1 inactivation during tumorigenesis, precancerous cells must accumulate additional genetic alterations, such as p53 mutations, but this requirement for an extra genetic "hit" contradicts the two-hit theory for the accelerated carcinogenesis associated with familial cancer syndromes. Here, we show that heterozygous BRCA1 inactivation results in genomic instability in nontumorigenic human breast epithelial cells in vitro and in vivo. Using somatic cell gene targeting, we demonstrated that a heterozygous BRCA1 185delAG mutation confers impaired homology-mediated DNA repair and hypersensitivity to genotoxic stress. Heterozygous mutant BRCA1 cell clones also showed a higher degree of gene copy number loss and loss of heterozygosity in SNP array analyses. In BRCA1 heterozygous clones and nontumorigenic breast epithelial tissues from BRCA mutation carriers, FISH revealed elevated genomic instability when compared with their respective controls. Thus, BRCA1 haploinsufficiency may accelerate hereditary breast carcinogenesis by facilitating additional genetic alterations.

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A PCR-based high-throughput screen with multiround sample pooling: application to somatic cell gene targeting

Cited by 22 publications

References 26 publications

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells

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