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. Author manuscript; available in PMC: 2013 Jan 10.
Published in final edited form as: Nat Cell Biol. 2011 Oct 23;13(11):1353–1360. doi: 10.1038/ncb2366

Fig. 1. Generation of mir-34a and mir-34b/c knockout MEFs.

Fig. 1

A. Diagrams of endogenous mir-34a and mir-34b/c gene structure and the knockout construct. Using recombineering, we engineered the mir-34a targeting vector with a ~6kb homologous arm on both 5’ and 3’ ends, flanking a Kozak sequence, a lacZ cDNA and a FRT-neo-FRT cassette. The mir-34b/c targeting vector contains a ~6kb homologous arm at each end, with the mir-34b/c gene and a Neo selection cassette flanked by loxP sites. b. Validating the germline transmission of the mir-34a and mir-34b/c targeted allele using Southern analysis. Putative WT, mir-34a+/- and mir-34a-/- animals derived from three independently targeted ES line were analyzed by Southern blot using probes either 5’ or 3’ to the homologous arms (left). Similar validation was performed for WT, mir-34b/c+/- and mir-34b/c-/- animals (right). c. Confirming loss of mir-34 expression in mir-34a and mir-34b/c knockout MEFs. Littermate-controlled WT, mir-34a+/- and mir-34a-/- MEFs were analyzed by real-time PCR to quantify the expression of mir-34a. While WT MEFs showed robust miR-34a induction upon culture stress, no miR-34a expression was detected in mir-34a-/- MEFs. The miR-34a level in mir-34a+/- MEFs was approximately half that of WT MEFs. Similar validation was performed for mir-34b/c-/- MEFs. Error bar, standard deviation, n=3.