1. Volume 6, Issue 6, Pages 1287-1295 (December 2000)
A Brg1 Null Mutation in the Mouse Reveals Functional Differences among Mammalian SWI/SNF Complexes 
Scott Bultman, Tom Gebuhr, Della Yee, Christian La Mantia, Jackie Nicholson, Anita Gilliam, Filippo Randazzo, Daniel Metzger, Pierre Chambon, Gerald Crabtree, Terry Magnuson 
Molecular Cell 
Volume 6, Issue 6, Pages (December 2000)
DOI: /S (00)

2. Figure 1 Targeted Disruption of the Mouse Brg1 Gene
(A) Schematic diagram of the wild-type locus, targeting vector, and mutant locus. Solid boxes represent Brg1 exons, the cross-hatched box depicts a promoterless neo-p(A) cassette, and the clear box represents plasmid vector sequence. Arrows 1–4 indicate the position and orientation of PCR primers used for genotype analysis in (G). A 3′ external probe used in (C) is shown as a solid bar beneath the wild-type locus. Restriction enzyme sites: RI, EcoRI; Xc , XcmI; X, XbaI; B, BstXI; S, SalI.
(B) Schematic diagram of the wild-type and truncated mutant proteins. Seven helicase motifs and a bromodomain are highlighted by vertical bars with roman numerals and a stippled box, respectively.
(C and D) Genomic Southern blot analysis of wild-type and heterozygous ES cell DNA. DNA samples were digested with BstXI (C) or XbaI (D) and hybridized with a 3′ external probe (C) or neo (D). Positions of wild-type and mutant fragments are indicated in kilobases.
(E) Northern blot analysis of total RNA from heterozygous ES cells hybridized with Brg1 cDNA (left) or neo (right) probes.
(F) Western blot analysis of nuclear extracts from wild-type and heterozygous ES cells utilizing antisera directed against the N-terminal portion of BRG1. The ∼200 kDa wild-type protein was detected in nuclei from cells of both genotypes. In contrast, the ∼125 kDa Brg1/Neo mutant protein could not be detected in heterozygous cells. Molecular weight standards are shown at the left in kilodaltons.
(G) Genotypic analysis of wild-type, heterozygous, and homozygous DNA by PCR. A 176 bp wild-type fragment is amplified by the 1-2 primer pair, and a 200 bp mutant fragment is amplified by the 3-4 primer pair. See (A) for the positions and orientations of the primers. MW, 1 kb plus molecular weight ladder.
Molecular Cell 2000 6, DOI: ( /S (00) )

3. Figure 2 Brg1 Heterozygous Phenotype
(A) Gross morphology of exencephalic (left) and normal (right) Brg1 heterozygotes at E17.5.
(B–D) H&E-stained tumor sections from Brg1 heterozygotes. (B) glandular structures of epithelial origin apparent at low magnification (100×). (C) Small, bland epithelial cells (arrowhead) are found in anastomosing cords (arrow) surrounding regions of central necrosis (*), giving rise to a variable cribiform pattern of growth. The stroma is also variable with hyalinized pink matrix (**), as well as pale amorphous regions (***) (400×). (D) Apocrine decapitation secretion is suggested in some areas (arrow) (400×).
Molecular Cell 2000 6, DOI: ( /S (00) )

4. Figure 3 In Vitro Blastocyst Outgrowths
Bright field photographs of heterozygous (A and C) or homozygous (B and D) Brg1 blastocysts after being cultured for 7 days in serum-supplemented media. Blastocysts were either placed directly into culture (A and B) or first treated with Pronase to remove zona pellucidae (C and D). (A) In the absence of Pronase, heterozygous explants hatched from the zona pellucidae (ZP), trophectoderm (TE) attached to and spread out across the bottom of tissue culture wells, and the inner cell masses (ICM) underwent extensive proliferation.
(B) In contrast, homozygotes failed to hatch and died. Pyknotic blastomeres are evident (arrow). (C) In the presence of Pronase, heterozygous explants exhibited robust outgrowth of the TE and ICM.
(D) In contrast, homozygotes attached to the bottom of the dish, but the TE and ICM died with little or no outgrowth. Pyknotic cells are evident (arrow).
Molecular Cell 2000 6, DOI: ( /S (00) )

5. Figure 4
Survival and Proliferation of Brg1-Deficient Primary Mouse Embryo Fibroblasts
Experimental (Exptl) and control (Cntrl) PMEFs have a Brg1 conditional mutation (Brg1L:La) balanced with the targeted null mutation (Brg1tm1Mag) or wild-type allele (Brg1+), respectively. Brg1L:La has two exons critical for function (solid boxes) that are flanked by loxP sites (solid triangles). Transfection of PMEFs with Cre (pNLS-Cre-EGFP) results in the two exons being excised and renders the cells homozyous (Exptl) or heterozygous (Cntrl). Cre-mediated deletions were confirmed by PCR utilizing primers (arrows) that amplify a 313 bp product. The primers are too far apart (2.2 kb) to amplify nondeleted DNA. Ethidium bromide–stained gels show the 313 bp PCR product specific for Cre-mediated deletion of Brg1L:La in two control (C) and two experimental (E) PMEF lines after being cultured for 3, 5, 7, or 9 days (D). Negative controls include DNA from untransfected Brg1L:La cells (N1), as well as wild-type mice (N2). MW, 1 kb plus molecular weight standard.
Molecular Cell 2000 6, DOI: ( /S (00) )

6. Figure 5
RT–PCR Expression Analysis of Brg1 and Brm in Preimplantation Mouse Embryos
Ethidium bromide–stained gels loaded with 585 bp Brg1 (A lanes) and 249 bp Brm (B lanes) PCR products. Each gel also includes the 1 kb plus molecular weight ladder at far left. Templates: 1, Brg1 cDNA plasmid (25 ng); 2, Brm cDNA plasmid (25 ng); 3, diluted Brg1 cDNA plasmid (100 fg); 4, diluted Brm cDNA plasmid (100 fg); 5, adult brain; 6, adult brain with enzyme omitted from reverse transcriptase reaction; 7, no template (i.e., water); 8, unfertilized eggs (25 per reaction); 9, two-cell embryos (51 per reaction); 10, four-cell embryos (16 per reaction); 11, morulae (27 per reaction); 12, blastocysts (34 per reaction); 13, feeder-free, undifferentiated ES cells.
Molecular Cell 2000 6, DOI: ( /S (00) )