Generation of inducible ES cells allowing temporal control of Smad1 depletion. (A) miSmad1abc-IRES-EGFP ES cells were generated by combining the transgenic technique reported by Kyba et al (2002)²⁴  with the miR-30–based shRNA technique reported by Sun et al (2006).²⁰  Three unique shRNA target sites were identified in the MH1 domain of Smad1, and hairpins were designed containing the miR-30 flanking sequence required for endogenous RISC processing. Cre/loxP-mediated recombination of a construct containing hairpins a, b, and c in tandem results in site-specific integration into a position downstream of the HPRT locus on the X chromosome, and confers neomycin resistance, allowing selection and isolation of transgenic cell clones amenable to tet-on induction of Smad1 knockdown, concomitant with GFP expression. (B) Genomic PCR analysis confirms single-copy site-specific integration of the shRNA-IRES-EGFP cassette. Two separate transgenic ES clonal cell lines are shown, 1A1 and 1D4. The parental AinV18 line serves as a negative control, and a transgenic iSmad1 clone that allows induced Smad1 expression¹⁶  provides a positive control. (C) GFP expression is seen in 1A1 and 1D4 ES cells 24 hours after induction of miSmad1abc hairpin expression, with parental AinV18 cells serving as negative control. (D) Analysis of metaphase karyotypes in the ES-cell lines, showing grossly normal chromosomal integrity in both clones, indistinguishable from parental AinV18 cells. (E) Representative RT-PCR analysis of Smad1 transcript levels in 2 clonal lines 30 hours after induction, showing a decrease in Smad1 transcript levels after addition of doxycycline, in contrast to unchanged levels of Smad5 transcripts, with Gapdh as a loading control. (F) Analysis of SMAD1 protein levels 30 hours after induction in 2 separate transgenic clonal lines by Western blotting. Overexpression of Smad1 using iSmad1 ES cells is used as a control to verify SMAD1 signal. (G) Densitometric analysis of SMAD1 protein levels normalized to the γ-tubulin loading control, demonstrating in a representative experiment the extensive knockdown (∼ 60%-90%) in both cell lines. (H) Representative Western blotting analysis of SMAD1 protein levels 48 hours after induction in embryoid bodies using 2 independent clonal cell lines (I) Densitometric quantification of Smad1 protein levels in a representative experiment, normalized to γ-tubulin controls, demonstrating approximately equivalent knockdown in 1A1 and 1D4 cell clones, with induction at either day 2 or day 4.