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Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis

Robert A. J. Oostendorp1,*, Alexander J. Medvinsky2,*, Nuray Kusadasi3, Naoki Nakayama4, Kirsty Harvey1, Claudia Orelio1, Katrin Ottersbach1, Todd Covey4, Rob E. Ploemacher3, Chris Saris4 and Elaine Dzierzak1,{ddagger}

1 Department of Cell Biology and Genetics, Erasmus University, Rotterdam, Netherlands
2 Centre for Genome Research, University of Edinburgh, Edinburgh, UK
3 Department of Hematology, Erasmus University, Rotterdam, Netherlands
4 Amgen, Thousand Oaks, CA, USA
* These authors contributed equally to this work



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  		Fig. 1. SV40 T antigen transgenic mice and stromal cell generation. (A) The promoters for the constitutively and ubiquitously expressed ß-actin and PGK genes were cloned 5' to the temperature-sensitive SV40 large T antigen (tsA58) gene. Transgenic mouse lines Tag5, containing one copy of the ß-actin tsA58 gene, and Tag11, containing one copy of the PGK tsA58 gene, were used to generate stromal cell lines and clones from embryonic tissues. (B) RT-PCR analysis of RNA from adult Tag5 and Tag11 mice show expression of the tsA58 transgene in all tissues tested. L, lymph nodes; K, kidney; S, spleen; BM, bone marrow, B, brain; M, muscle. (C) The embryonal subregions used for the isolation of stromal clones. E10 and E11 mouse embryos transgenic for the tsA58 gene or a lacZ marker gene were used to generate stromal cell lines and clones from dorsal aorta and mesenchyme (AM), urogenital ridges (UG, mesonephros and genital ridges), the gastrointestinal region (GI) and the liver (EL). Under our culture conditions, no cell lines could be established from the yolk sac. Numbers in brackets indicate the number of clones isolated from the various subregions.

 


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  		Fig. 2. Growth rate analysis of cell lines established from midgestation embryos. Cell lines were established from the (A) aorta-mesenchyme, (B) urogenital ridges, (C) embryonic liver and (D) gastrointestinal tissues of embryonic day 10 and 11 mice. The number of cells in each culture was counted at each passage, and the total number of viable cells (calculated) is plotted against time (weeks in culture after initial plating). Black-filled symbols represent cell lines derived from the tsA58 transgenic embryos, and white-filled symbols represent cell lines derived from the control lacZ transgenic embryos. All cell lines and clones established from Tag5 and Tag11 transgenic embryos contained the tsA58 transgene as determined by PCR analysis.

 




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