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| Caption | A) Targeting strategy used to disrupt the Hsd11b1 (11betaHSD1) locus. Homologous recombination (represented by X) between the targeting vector and the Hsd11b1 gene results in the replacement of exons 4 and 5 with the selection cassette. The Hsd11b1 targeting vector was derived using the Lambda KOS system. The Lambda KOS phage library, arrayed into 96 superpools, was screened by PCR using exons 4 and 5-specific primers Hsd-1 (5'-AGGTAGTGTCTCGCTGCCTT-3') and Hsd-3 (5'-CTTCGCACAGAGTGGATGTC-3'). PCR-positivephage superpools were plated and screened by filter hybridization using the 306 bp amplicon derived from primers Hsd-1 and Hsd-3 as a probe. Three pKOS genomic clones, pKOS-63, pKOS-73 and pKOS-82, were isolated from the library screen and confirmed by sequence and restriction analysis. The yeast selection cassette and pKOS-73 were co-transformed into yeast. Clones that had undergone homologous recombination to replace a 408 bp region containing exon 4 and exon 5 with the yeast selection cassette were isolated. The yeast cassette was subsequently replaced with a LacZ/Neo selection cassette to complete the Hsd11b1 targeting vector. The Not I linearized targeting vector was electroporated into 129S5/SvEvBrd (Lex-1) ES cells. G418/FIAU resistant ES cell clones were isolated, and correctly targeted clones were identified and confirmed by Southern analysis using a 328 bp 5' external probe (9/10), generated by PCR using primers Hsd-9 (5'-CAATGCATCCATGCGCCTGAA-3') and Hsd-10 (5'-AGAGACCAGACATTAGGACAC-3') and a 607 bp 3' internal probe (Neo5/2), amplified by PCR using primers Neo-5 (5'-GGCAGCGCGGCTATCGTG-3') and Neo-2 (5'-TCAGAAGAACTCGTCAAG-3'). | ||||
| Copyright | This image is from Garcia RA, PLoS One 2013;8(2):e53192, and is displayed under the terms of the Creative Commons Attribution 4.0 International License. J:199430 | ||||
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Associated Alleles |
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Mouse Genome Database (MGD), Gene Expression Database (GXD), Mouse Models of Human Cancer database (MMHCdb) (formerly Mouse Tumor Biology (MTB)), Gene Ontology (GO) |
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last database update 05/07/2024 MGI 6.23 |
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