[CITATION][C] Generation of Smad4/Dpc4 conditional knockout mice

X Yang, C Li, PL Herrera, CX Deng - genesis, 2002 - Wiley Online Library
genesis, 2002Wiley Online Library
Smad4/Dpc4 is a central mediator of TGF-β signals (Massague, 1998). It was previously
shown that loss of Smad4 results in lethality at embryonic (E) days 6–7 due to impaired
extraembroynic membrane formation and decreased epiblast proliferation (Sirard et al.,
1998; Yang et al., 1998). To overcome the early lethality and to study functions of Smad4 at
later stages, we generated a Smad4 conditional knockout allele using the Cre-loxP
approach (Nagy, 2000). Smad4 was targeted by ploxPneoSmad4, a cotransfertype targeting …
Smad4/Dpc4 is a central mediator of TGF-β signals (Massague, 1998). It was previously shown that loss of Smad4 results in lethality at embryonic (E) days 6–7 due to impaired extraembroynic membrane formation and decreased epiblast proliferation (Sirard et al., 1998; Yang et al., 1998). To overcome the early lethality and to study functions of Smad4 at later stages, we generated a Smad4 conditional knockout allele using the Cre-loxP approach (Nagy, 2000).
Smad4 was targeted by ploxPneoSmad4, a cotransfertype targeting vector (Deng et al., 1993), which introduced a ploxPneo cassette (Yang et al., 1998) and a third loxP into intron 8 and 7 of the Smad4 locus, respectively (Fig. 1A). The targeted ES cell clones were identified by Southern blot analysis using both flanking and internal probes (Fig. 1B). Chimeras were generated by injections of targeted ES cells into C57BL/6 blastocysts. After germline transmission by crosses with Black Swiss mice, we found that the presence of the ploxPneo in intron 8 completely blocked normal splicing of Smad4 and caused recessive lethality at around E7 (not shown), a phenotype resembling that of Smad4-null embryos (Yang et al., 1998). Therefore, we removed the plox-Pneo cassette from the germ line by crossing the het-
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