Na$ sp+$-Pi cotransport across the brush border membrane is the rate limiting step in renal Pi reabsorption. To determine its precise role in the maintenance of Pi homeostasis, we cloned and characterized the renal-specific Na$ sp+$-Pi cotransporter/Npt2 gene and generated a gene targeting vector for the creation of a knockout mouse. The gene was cloned by screening a genomic DNA library with a rat Npt2 cDNA probe. The Npt2 gene is approximately 17kb and contains 13 exons and 12 introns. A targeting construct was generated by inserting 5$ sp prime$ and 3$ sp prime$ homologous arms of 2.1 and 2kb, respectively, into the pPNT vector and replacing 7.7kb of Npt2 with a neomycin resistance gene. The vector also contained the herpes simplex virus thymidine kinase gene for negative selection. After electroporation into embryonic stem cells, clones were picked following selection in G418 and FIAU. Of 100 doubly-resistant clones that were screened by Southern analysis, 6 positive clones were detected giving a targeting frequency of 6%.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.23894 |
| Date | January 1996 |
| Creators | Hewson, A. Stacy (Allison Stacy) |
| Contributors | Tenenhouse, H. S. (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001508073, proquestno: MM12203, Theses scanned by UMI/ProQuest. |
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