Cre-mediated heterozygous deletion of Med28 causes differentiation of iPSC.

Abstract

<p><b>(A)</b><i>Med28</i>^<i>fl/fl</i> iPSCs were infected with Ad-Cre, followed by single cell plating on day 3 and genotyping along with other analyses performed between days 5–8. <b>(B)</b> PCR genotyping shows clones with both deleted (- allele) and non-deleted WT (+ allele) alleles infected with Ad-Cre (+ Cre) compared to uninfected clones (-Cre), respectively. <b>(C)</b> Representative bright field images show noticeable differences in morphology between non-deleted (panel A, -Cre) and deleted (panel B, +Cre) <i>Med28</i>^<i>fl/fl</i> iPSC colonies (scale bar 200μm). Note the differentiated colony morphology of the <i>Med28</i>^<i>fl/fl</i> (panel B, +Cre) cells. <b>(D)</b> Quantitation of real-time PCR for deleted <i>Med28</i>^<i>fl/fl</i> iPSC colonies (+Cre) show ~2-fold decreased expression of <i>Med28</i> as well as significantly lower expression of <i>Oct4</i> and <i>Nanog</i> (n = 4; *, p<0.05) compared to non-deleted <i>Med28</i>^<i>fl/fl</i> iPSC (-Cre) colonies. <b>(E)</b> Immunostaining of deleted <i>Med28</i>^<i>fl/fl</i> colonies (panels E-H) show loss of self-renewal factor Oct4 (red, C and G) and Nanog (green, D and H) compared to non-deleted colonies (panels A-D). Bright field images (A and E) and nuclear DAPI (blue, B and F) are also shown (scale bar 200μm). <b>(F)</b> Quantitation of real—time PCR for differentiation markers reveal increased differentiation markers in deleted (+Cre) <i>Med28</i>^<i>fl/fl</i> iPSC colonies for extra embryonic lineage (<i>Gata4</i>, <i>Hand1</i>, and <i>Cdx2</i>) and decreased germ layer lineage makers nestin (Nes, ectoderm) and T brachyury (T, mesoderm) compared to non-deleted (-Cre) <i>Med28</i>^<i>fl/fl</i> iPSCs colonies (n = 4). Data are presented as mean +/- SD (*<i>p</i><0.05).</p