Xin et al. 10.1073/pnas.0604604103. |
Supporting Figure 6
Supporting Table 1
Supporting Materials and Methods
Fig. 6. Generation of GATA6 knockout mice. (A) A diagram of the targeting strategy for the GATA6 locus is shown. A neomycin resistance cassette replacing »1 kb of the GATA6 gene encoding amino acids 1-372 is depicted. The structure of the targeted allele and predicted genomic structure after homologous recombination, resulting in deletion of exon 2, is shown. (B) Southern blot analysis of genomic DNA from mice of the indicated genotypes demonstrating the 6.0-kb WT and 3.5-kb mutant HindIII fragments. A 600-bp DNA fragment upstream of the 5' arm was used to probe HindIII-digested tail DNA. The position of the primers and strategy for PCR genotyping are also shown. H, HindIII site used for Southern blot analyses. (C) PCR genotyping of GATA6 mice showing the 150-bp WT and the 430-bp mutant products using the primers shown in A. A 600-bp DNA fragment upstream of the 5' arm was used to probe HindIII-digested tail DNA. The position of the primers and strategy for PCR genotyping are also shown. The HindIII site used for Southern blot analyses is also depicted.
Table 1. Genotypes of offspring from GATA4+/-, GATA6+/- intercrosses
Offspring of each genotype | ||||
Age | +/+ | GATA4+/- | GATA6+/- | GATA4+/-/6+/- |
P1 | 29 (36%) | 27 (34%) | 24 (30%) | 0 (0%) |
E14.5 | 8 (30%) | 9 (33%) | 10 (37%) | 0 (0%) |
E10.5-13.5 | 26 (26%) | 28 (28%) | 26 (26%) | 19 (19%) |
GATA4+/-
mice were mated with GATA6+/- mice. Numbers of viable offspring of each genotype and percentage of total (in parentheses) at each developmental stage are shown. P1, postnatal day 1.Supporting Materials and Methods
Mice. Heterozygous GATA6+/- mice were maintained in both a pure SV129 and a mixed background of SV129 and C57BL6. Heterozygous GATA4+/- (1) and GATA6+/- mice of mixed background were intercrossed, and the appearance of the vaginal plug was taken as E0.5. Genomic DNA was isolated from embryos, and genotypes of fetuses were determined by PCR. Primer sequences are available on request. For the GATA6 genotyping, 150- and 430-bp PCR fragments were obtained from WT and GATA6 homozygous mutant animals, respectively. SM22-lacZ transgenic mice are described in ref. 2.
Histology and Immunostaining.
Embryos were collected at various time points of gestation from E10.5 to E14.5 and fixed in 4% paraformaldehyde in PBS at 4°C overnight followed by dehydration and embedding in paraffin as described in ref. 3. Sections of 5 mm thickness were mounted on slides, followed by H&E staining according to standard procedures. For immunostaining, sections were deparaffinized in xylene, rehydrated through graded ethanol to PBS, and permeabilized in 0.3% Triton X-100 in PBS. Sections were then blocked by 1.5% normal horse serum in PBS followed by incubation with anti-smooth muscle a-actin clone 1A4 (Sigma-Aldrich) or rabbit anti-phosphohistone H3 (Upstate Cell Signaling Solutions, Charlottesville, VA) at a 1:200 dilution in 0.1% BSA in PBS overnight at 4°C. Sections were washed in PBS, and Cy3 or fluorescein-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA) were applied at a 1:200 dilution in 1% normal horse serum for 1 h. For the phosphohistone H3 studies, the measurements represent the average of six sections of control and mutant embryonic hearts with standard deviations.Whole-Mount Staining for b-Gal.
Embryos were collected at E12.0 and stained for b-gal as described in ref. 4. After overnight staining, embryos were fixed in 4% paraformaldehyde/0.2% glutaraldehyde, dehydrated through graded methanol, and cleared in benzyl benzoate:benzyl alcohol (2:1).Whole-Mount Immunostaining.
E10.5 embryos were fixed in 4% paraformaldehyde/PBS overnight at 4°C and bleached with 5% H2O2 for 4 h to block endogenous peroxidase. Embryos were blocked with PBSMT (3% milk/0.1%Triton X-100 in PBS) for 2 h at room temperature, followed by overnight incubation with 10 mg/ml anti-PECAM (BD Biosciences, San Jose, CA) at 4°C. Embryos were subsequently washed in PBSMT and incubated with 1:100 dilution of anti-rat HRP-coupled secondary antibody at 4°C overnight. Staining was visualized by using 0.3 mg/ml 3,3¢-diaminobenzidine (Sigma-Aldrich), 0.5% NiCl2, and 0.03% H2O2.1. Molkentin, J. D., Lin, Q., Duncan, S. A. & Olson, E. N. (1997) Genes Dev. 11, 1061-1072.
2. Li, L., Miano, J. M., Mercer, B. & Olson, E. N. (1996) J. Cell Biol. 132, 849-859.
3. Moller, W. & Moller, G. (1994) Biotech. Histochem. 69, 289-290.
4. Naya, F. J., Wu, C., Richardson, J. A., Overbeek, P. & Olson, E. N. (1999) Development (Cambridge, U.K.) 126, 2045-2052.