A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Export

@article{citeulike:1678628, abstract = {Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.}, address = {Mouse Cancer Genetics Program, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA.}, author = {Lee, E. C. and Yu, D. and Martinez de Velasco, J. and Tessarollo, L. and Swing, D. A. and Court, D. L. and Jenkins, N. A. and Copeland, N. G.}, citeulike-article-id = {1678628}, citeulike-linkout-0 = {http://dx.doi.org/10.1006/geno.2000.6451}, citeulike-linkout-1 = {http://linkinghub.elsevier.com/retrieve/pii/S0888-7543(00)96451-6}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/11352566}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=11352566}, day = {1}, doi = {10.1006/geno.2000.6451}, issn = {0888-7543}, journal = {Genomics}, keywords = {2001, bac, cloning, generating, mouse, mtlur5, tian}, month = {April}, number = {1}, pages = {56--65}, posted-at = {2008-10-14 07:17:14}, priority = {2}, title = {A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.}, url = {http://dx.doi.org/10.1006/geno.2000.6451}, volume = {73}, year = {2001} }

TY - JOUR ID - citeulike:1678628 L3 - citeulike-article-id:1678628 N2 - Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era. IS - 1 JF - Genomics SN - 0888-7543 AD - Mouse Cancer Genetics Program, National Cancer Institute-Frederick, Frederick, Maryland 21702, USA. EP - 65 TI - A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. VL - 73 SP - 56 KW - 2001 KW - bac KW - cloning KW - generating KW - mouse KW - mtlur5 KW - tian AU - Lee, EC AU - Yu, D AU - Martinez de Velasco, J AU - Tessarollo, L AU - Swing, DA AU - Court, DL AU - Jenkins, NA AU - Copeland, NG PY - 2001/04/01/ UR - http://dx.doi.org/10.1006/geno.2000.6451 ER -