Supplementary Figure 7: Generation of Smarcd2-knockout mice.

(a) Mouse Smarcd2 (ENSMUST00000021052)-mutant embryonic stem cells (ES cells with heterozygous deletion of Smarcd2; clone AF4) were purchased from KOMP repository. Shown is Smarcd2^{tm1(KOMP)Vlcg} (deletion allele). The positions of the primer pairs for the wild-type allele (empty arrowheads) and the knockout allele (filled arrowheads) are shown in relation to the Smarcd2 gene and deletion allele, respectively. (b) PCR-based genotyping. F₁ intercrosses of heterozygous mice resulted in Smarcd2^+/+, Smarcd2^+/−, and Smarcd2^−/− embryos. PCR was performed according to the manufacturer (KOMP repository): wild-type allele, 264 bp (empty arrowheads) and knockout allele, 569 bp (filled arrowheads). Embryos 13, 15, and 17 are of the knockout genotype (Smarcd2^−/−), and embryos 12, 14, and 16 are of the heterozygous genotype (Smarcd2^+/−). Images have been cropped; full-length images are shown in Supplementary Data 3d,e. (c–e) Mendelian inheritance pattern of mutant Smarcd2 alleles. (c) Knockout embryos are not viable after birth. The numbers of Smarcd2^+/+ (n = 24), Smarcd2^+/− (n = 52), and Smarcd2^−/− (n = 1, perinatal death) offspring born are displayed. (d) The distributions of genotypes per litter (in percent) are displayed. In total, expected Mendelian ratios of the genotypes (percent of genotype per litter) are observed in utero. Data from 15 litters are shown; compare with Supplementary Table 20. Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. (e) The number of embryos per genotype and gestational age are shown at 12.5, 13.5, 14.5, 15.5, and 16.5 d.p.c. In total, wild type n = 24, heterozygous n = 56, and knockout n = 27 embryos were observed. (f) Western blot analysis detecting members of the SWI/SNF complex in Smarcd2-knockout embryos. Protein expression of SMARCD1 (BAF60a), SMARCD2 (BAF60b), SMARCD2 (BAF60c) (kindly provided by J. Lessard, Montreal), and SWI/SNF members BRG1, BAF170, BAF155, and BAF47 are shown. Images of membranes have been cropped; full images are available in Supplementary Data 4. Protein expression was determined in crude embryonic tissue lysates by western blotting. Embryos from two litters were analyzed: wild type n = 3, heterozygous n = 3, knockout n = 3. Replicates: 1. (g) Enumeration of total white blood cells (CD45.2⁺) and the stem cell compartment (LSK cells and LSK CD150⁺CD48⁻ cells) per embryo in wild-type, heterozygous, and knockout mouse fetal liver hematopoiesis. Smarcd2^−/− embryos show reduced CD45.2⁺ blood cells but comparable amounts of stem cells (LSK, LSK CD150⁺CD48⁻). Data are from three litters: wild type n = 7; heterozygous n = 10; knockout n = 8. Center value, mean; error bars, s.e.m. P values were calculated by two-tailed unpaired t test. Replicates: 1.