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| Caption | Supplemental Fig. 1. Construction of the Extl2 targeting vector to generate the Extl2tm1Hkit allele. Step 1: The genomic clone spanning exons 3 through 5 of the Extl2 gene was digested with NotI and XhoI, and the resulting exon-containing fragment was inserted into pBluescript II KS (Stratagene). Step 2: The neomycin cassette was amplified via PCR; the PCR mixture included the pPGK-NeobpA vector as a template and the following primers: sense primer, 5' - CCCAAGCTTCTACCGGGTAGGGGAG-3' (underline denotes a HindIII site); antisense primer, 5' - CGGAATTCCCCAGCTGGTTCTTCCG-3' (underline denotes an EcoRI site). The resulting PCR fragments were ligated onto the DNA fragment that was generated by digestion of pBluescript II KS-EXTL2 with HindIII and EcoRI. Integrity of pBluescript II KS-EXTL2-Neo was confirmed by sequencing the entire coding region of the Neo gene and the ligation junction. The Neo gene was inserted in an antiparallel orientation; consequenctly, exons 3 and 4 of Extl2 were disrupted. Step 3: A diphtheria toxin selection gene (DT-A) fragment obtained from digestion of the pMC1-DTpA vector with XhoI and BglII was ligated onto a linear pcDNA3.1/Zeo(-) (Invitrogen) that had been digested with XhoI and BamHI. The DNA fragment containing the DT-A gene that was generated by digestion of pcDNA3.1/Zeo(-)-DT-A with XhoI and KpnI was inserted into pBluescript II KS. Step 4: The pBluescript II KS-EXTL2-Neo vector prepared in step 2 was digested with NotI and XhoI, and the resulting DNA fragment that contained exons 3 and 5 of Extl2 and the Neo gene was inserted between the NotI and XhoI sites of the pBluescript II KS-DT-A prepared in step 3. Step 5: A DNA fragment containing Extl2 exon 5 that had been generated by digestion of pBK-RSV-EXTL2 with XhoI was inserted into the XhoI site of pBluescript II KS-EXTL2-Neo-DT-A, which had been prepared in step 4. | ||||
| Copyright | This image is from Nadanaka S, J Biol Chem 2013 Mar 29;288(13):9321-33 and is displayed with the permission of the American Society for Biochemistry and Molecular Biology who owns the Copyright. Full text from JBC. J:197845 | ||||
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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 04/23/2024 MGI 6.23 |
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