Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines. Export

@article{citeulike:953409, abstract = {Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian--particularly human--genes.}, address = {Yokohama City University, Yokohama, Japan.}, author = {Iiizumi, S. and Nomura, Y. and So, S. and Uegaki, K. and Aoki, K. and Shibahara, K. and Adachi, N. and Koyama, H.}, citeulike-article-id = {953409}, citeulike-linkout-0 = {http://view.ncbi.nlm.nih.gov/pubmed/16989091}, citeulike-linkout-1 = {http://www.hubmed.org/display.cgi?uids=16989091}, issn = {0736-6205}, journal = {Biotechniques}, keywords = {cells, es, gene, method, targeting}, month = {September}, number = {3}, pages = {311--316}, posted-at = {2006-11-20 16:03:20}, priority = {3}, title = {Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines.}, url = {http://view.ncbi.nlm.nih.gov/pubmed/16989091}, volume = {41}, year = {2006} }

TY - JOUR ID - citeulike:953409 L3 - citeulike-article-id:953409 N2 - Targeted gene disruption is a powerful tool for studying gene function in cells and animals. In addition, this technology includes a potential to correct disease-causing mutations. However, constructing targeting vectors is a laborious step in the gene-targeting strategy, even apart from the low efficiency of homologous recombination in mammals. Here, we introduce a quick and simplified method to construct targeting vectors. This method is based on the commercially available MultiSite Gateway technology. The sole critical step is to design primers to PCR amplify genomic fragments for homologous DNA arms, after which neither ligation reaction nor extensive restriction mapping is necessary at all. The method therefore is readily applicable to embryonic stem (ES) cell studies as well as all organisms whose genome has been sequenced. Recently, we and others have shown that the human pre-B cell line Nalm-6 allows for high-efficiency gene targeting. The combination of the simplified vector construction system and the high-efficiency gene targeting in the Nalm-6 cell line has enabled rapid disruption of virtually any locus of the human genome within one month, and homozygous knockout clones lacking a human gene of interest can be created within 2-3 months. Thus, our system greatly facilitates reverse genetic studies of mammalian--particularly human--genes. IS - 3 JF - Biotechniques SN - 0736-6205 AD - Yokohama City University, Yokohama, Japan. EP - 316 TI - Simple one-week method to construct gene-targeting vectors: application to production of human knockout cell lines. VL - 41 SP - 311 KW - cells KW - es KW - gene KW - method KW - targeting AU - Iiizumi, S AU - Nomura, Y AU - So, S AU - Uegaki, K AU - Aoki, K AU - Shibahara, K AU - Adachi, N AU - Koyama, H PY - 2006/09// UR - http://view.ncbi.nlm.nih.gov/pubmed/16989091 ER -