PURPOSE: Our understanding of urogenital tract development and its response to disease or injury is hindered by complex interactions between epithelial and mesenchymal cells, and the difficulties in studying either component in isolation. We investigated whether transgenic mice could be generated to express enhanced green fluorescent protein (EGFP) in smooth muscle cells (SMCs) and whether such cells could then be purified using flow cytometric sorting to isolate RNA to be used in future gene expression assays. MATERIALS AND METHODS: A 13.7 kb mouse smooth muscle gamma-actin promoter fragment was ligated to an EGFP reporter gene and microinjected into male mouse pronuclei. Adult transgenic mice were sacrificed and urogenital tissues were removed for histological and immunohistochemical studies. In other animals conditions were determined for dissociating bladder cells and the subsequent purification of bladder SMCs by sorting. RESULTS: Six lines of transgenic mice were generated (transgene copy numbers 1 to 30). EGFP was expressed in all smooth muscle beds examined except those associated with small blood vessels. EGFP levels appeared to correlate with transgene copy number. Histological and immunohistochemical analysis confirmed that reporter gene expression was restricted to SMCs of all tissues examined. Parameters for generating bladder cell suspensions were established and EGFP labeled bladder SMCs were identified by flow cytometric analysis. CONCLUSIONS: Several lines of transgenic mice have been generated in which SMCs of urogenital tissues have been labeled with EGFP and pure populations of SMCs have been obtained. The methods established for the rapid dissociation and purification of bladder SMCs should minimize degradative changes. These approaches may enable us to address issues involving bladder SMC development and differentiation as well as the response to injury and disease by performing transcriptome wide analyses on purified SMC populations.
PURPOSE: Our understanding of urogenital tract development and its response to disease or injury is hindered by complex interactions between epithelial and mesenchymal cells, and the difficulties in studying either component in isolation. We investigated whether transgenic mice could be generated to express enhanced green fluorescent protein (EGFP) in smooth muscle cells (SMCs) and whether such cells could then be purified using flow cytometric sorting to isolate RNA to be used in future gene expression assays. MATERIALS AND METHODS: A 13.7 kb mousesmooth muscle gamma-actin promoter fragment was ligated to an EGFP reporter gene and microinjected into male mouse pronuclei. Adult transgenic mice were sacrificed and urogenital tissues were removed for histological and immunohistochemical studies. In other animals conditions were determined for dissociating bladder cells and the subsequent purification of bladder SMCs by sorting. RESULTS: Six lines of transgenic mice were generated (transgene copy numbers 1 to 30). EGFP was expressed in all smooth muscle beds examined except those associated with small blood vessels. EGFP levels appeared to correlate with transgene copy number. Histological and immunohistochemical analysis confirmed that reporter gene expression was restricted to SMCs of all tissues examined. Parameters for generating bladder cell suspensions were established and EGFP labeled bladder SMCs were identified by flow cytometric analysis. CONCLUSIONS: Several lines of transgenic mice have been generated in which SMCs of urogenital tissues have been labeled with EGFP and pure populations of SMCs have been obtained. The methods established for the rapid dissociation and purification of bladder SMCs should minimize degradative changes. These approaches may enable us to address issues involving bladder SMC development and differentiation as well as the response to injury and disease by performing transcriptome wide analyses on purified SMC populations.
Authors: Han Sheng Chiu; John C Szucsik; Kylie M Georgas; Julia L Jones; Bree A Rumballe; Dave Tang; Sean M Grimmond; Alfor G Lewis; Bruce J Aronow; James L Lessard; Melissa H Little Journal: Dev Biol Date: 2010-05-24 Impact factor: 3.582
Authors: Debashish Roy; Grant J Steyer; Madhusudhana Gargesha; Meredith E Stone; David L Wilson Journal: Anat Rec (Hoboken) Date: 2009-03 Impact factor: 2.064