Establishment of embryonic kidney and facial culture systems to evaluate the role of the transcription factor Six2 through RNAi

The Br mouse mutation results in frontonasal and renal hypodysplasia. Linkage analysis mapped the Br mutation close to the homeobox transcription factor Six2 , which is normally expressed in the metanephric and facial mesenchyme during embryonic development. The purpose of this study is to evaluate Six2 's role in nephrogenesis and craniofacial morphogenesis by silencing Six2 in embryonic kidney and facial prominence organ cultures. Kidney organ cultures were established from E11.5 mouse embryos, cultured for 48 hours, and immunostained to reveal ureteric bud branching patterns. Frontonasal (FNP), lateral nasal (LNP), and maxillary prominences (MXP) of E11.5 embryos were dissected and Six2 expression was measured with qRT‐PCR. Six2 expression was highest in the FNP, 3‐fold less in the MXP, and 6‐fold less in the LNP. This was confirmed by immunostaining in embryos at the same age. We evaluated five different shRNA constructs for silencing efficiency of Six2 using p19 embryonal carcinoma cells. As measured with qRT‐PCR, Six2 expression was unaffected using four of the constructs and decreased expression by 30–50% with the fifth. Using this shRNA construct, we will transfect our established culture systems, confirm Six2 expression is down‐regulated, and then evaluate differences in kidney tubule branching and FNP cell proliferation.