Andy McMahon at Harvard University and Gli1 a kind gift from Dr Alexandra

Andy mcmahon at harvard university and gli1 a kind

This preview shows page 2 - 4 out of 8 pages.

Andy McMahon at Harvard University), and Gli1 (a kind gift from Dr. Alexandra Joyner, Sloan Kettering Institute, New York). Details of the in situ analysis protocol may be obtained upon request. Results Generation of a Tcf21-Cre founder mouse line The targeting construct for Tcf21-Cre was designed to replace the first exon of the Tcf21 gene with Cre recombinase and a neomycin cassette (Fig. 1A). After electroporation, murine ES cell clones were screened by Southern blot analysis using a HindIII digest and 3 9 probe outside the region of homology. One correctly targeted ES cell clone was used to generate chimeric mice that gave germline transmission (Fig. 1B). Tcf21-Cre driver mouse line excises genes in the metanephric mesenchyme and its derivatives Tcf21-Cre mice were bred with Z/EG reporter mice. In embryonic day 10.5 metanephros, expression of the GFP reporter (denoting successful Cre-mediated excision) was observed in the condensing metanephric mesenchyme surrounding the invading ureteric bud (Fig. 1C). At this stage, GFP staining appeared to be thin and mosaic. At E11.5, GFP expression in metanephric Generation of Tcf21/Pod1-Cre Mouse PLoS ONE | 2 July 2012 | Volume 7 | Issue 7 | e40547
Image of page 2
mesenchyme was evident (Fig. 1D). By E13.5, gene deletion activity was shown in broad areas in the mesenchyme including condensing mesenchyme, developing nephrons such as pretubular aggregates and comma-shaped bodies, interstitial stromal cells, and the mesenchyme surrounding the stalk of ureteric bud (Fig. 1E). At E16.5, in addition to the GFP expression observed at E13.5, GFP expression was also appreciated in S-shaped bodies, immature podocytes in capillary loop glomeruli, and immature tubular cells (Fig. 1F). We compared the domain of Tcf21 -driven Cre excision to that of the Six2-Cre mice. As seen in Fig. 1G, Six2- Cre activity is restricted to cap mesenchyme and its derivatives. At postnatal day 0, in Tcf21-Cre;Z/EG mice, GFP was observed in podocytes, tubules and cells of Bowman’s capsule, though it appeared mosaic in the tubules (Fig. 1H). Double immunostaining for GFP and pancytokeratin showed no overlap, demonstrating that Tcf21-Cre is not active in collecting duct cells that are derived from the ureteric bud lineage (Fig. 1I). Taken together, Tcf21-Cre has the capacity for broad gene deletion in the mesenchyme and its derivatives, including both interstitial cells and epithelial components of the nephron. Thus, Tcf21-Cre is a valuable resource permitting the use of a single mouse Cre-driver strain to gain insight into multiple metanephric populations. Tcf21-Cre excises genes in mesenchymes in multiple organs To explore the full potential of the Tcf21-Cre line, extrarenal Cre activity was assessed. At E10.5, GFP expression was observed in the 1 st to 3 rd branchial arches (Fig. 2 AB), the heart, and in metanephric mesenchyme surrounding the ureteric bud (Fig. 2C and 1B). E11.5 embryos showed a similar pattern (data not shown). At E13.5 and E16.5, GFP expression was prominent in epicardium, lung mesenchyme, kidney mesenchyme, lamina
Image of page 3
Image of page 4

You've reached the end of your free preview.

Want to read all 8 pages?

  • Fall '20
  • john
  • The Land, Nephron, PLOS One, mesenchyme, hydroureter

What students are saying

  • Left Quote Icon

    As a current student on this bumpy collegiate pathway, I stumbled upon Course Hero, where I can find study resources for nearly all my courses, get online help from tutors 24/7, and even share my old projects, papers, and lecture notes with other students.

    Student Picture

    Kiran Temple University Fox School of Business ‘17, Course Hero Intern

  • Left Quote Icon

    I cannot even describe how much Course Hero helped me this summer. It’s truly become something I can always rely on and help me. In the end, I was not only able to survive summer classes, but I was able to thrive thanks to Course Hero.

    Student Picture

    Dana University of Pennsylvania ‘17, Course Hero Intern

  • Left Quote Icon

    The ability to access any university’s resources through Course Hero proved invaluable in my case. I was behind on Tulane coursework and actually used UCLA’s materials to help me move forward and get everything together on time.

    Student Picture

    Jill Tulane University ‘16, Course Hero Intern

Stuck? We have tutors online 24/7 who can help you get unstuck.
A+ icon
Ask Expert Tutors You can ask You can ask You can ask (will expire )
Answers in as fast as 15 minutes
A+ icon
Ask Expert Tutors