1. Volume 133, Issue 6, Pages 1948-1959 (December 2007)
Severe Intestinal Obstruction on Induced Smooth Muscle–Specific Ablation of the Transcription Factor SRF in Adult Mice 
Meike Angstenberger, Jörg W. Wegener, Bernd J. Pichler, Martin S. Judenhofer, Susanne Feil, Siegfried Alberti, Robert Feil, Alfred Nordheim 
Gastroenterology 
Volume 133, Issue 6, Pages (December 2007)
DOI: /j.gastro
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2. Figure 1
Generation of SMC-specific SRF knockout mice. (A) Genotyping by PCR of different organs of tamoxifen- or solvent-treated mice. Eight-week-old Srfflex1/flex1:SM-CreERT2/wt mice were treated with 1 mg of tamoxifen (or solvent) for 5 consecutive days and killed 7 days after the last injection, and genomic DNA was isolated. Cre-mediated recombination of the Srfflex1 allele is mainly restricted to adult organs containing a high percentage of SM tissue. (B) Western blot analysis of SRF levels in different organs. Protein extracts were prepared from the same mice used in panel A. Full-length SRF protein was significantly reduced in organs containing a high percentage of SM, whereas non-SM tissue showed similar levels of SRF protein in control and mutant mice. The * indicates a currently nonidentified protein signal. Comparable loading of protein is confirmed by reprobing the membrane with anti-GAPDH. (C) Quantification of full-length SRF protein levels in control (n = 4) and mutant (n = 4) mice using ImageQuant 5.1 (Molecular Dynamics) software. Protein levels of control mice were set to 100%. Each column of mutant organs represents the mean of 4 different mutant mice. Error bars correspond to standard deviation (±SD). (**P < .01, ***P < .001, Student t test). (D) Anti-SRF immunostaining of colon tissue of tamoxifen-treated Srfflex1/flex1:SM-CreERT2/wt (mutant) and Srfflex1/flex1:wt/wt (control) 9-week-old mice. Colon preparations were isolated 6 days after the last tamoxifen injection. Control animals display strong nuclear staining of SRF protein (indicated by arrows). (Scale bars, 50 μm).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
Phenotypic analysis of SM-specific SRF knockout mice. (A) Survival curves of mutant and control mice, showing 100% lethality of the SMC-specific SRF knockout mice within 11–16 days after the first injection of tamoxifen. All analyzed 6- to 12-week-old mice were given a daily intraperitoneal injection of 1 mg of tamoxifen or solvent on 5 consecutive days (arrows). Mutant mice (n = 29) were Srfflex1/flex1:SM-CreERT2/wt treated with tamoxifen; whereas the control group (n = 27) was composed of either Srfflex1/flex1:SM-CreERT2/wt mice treated with solvent, Srfflex1/wt:SM-CreERT2/wt mice, or Srfflex1/flex1:wt/wt mice treated with tamoxifen. All mutant mice showed the severe intestinal phenotype shown in panel B. (B) Macroscopic analysis of the GI tract of control and mutant mice. The SM-specific SRF knockout mice displayed massively dilated, obstructed caecum (arrow) and colon (arrowhead) within 3–11 days after the last injection.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Analysis of intestinal transit using x-ray computer tomography (CT). SRF deficiency in adult SM results in hampered intestinal transit. Twelve-week-old Srfflex1/wt:SMCreERT2/wt (control) (A–E) and Srfflex1/flex1:SMCreERT2/wt (mutant) (F–J) mice were analyzed 5 days after the last tamoxifen (or solvent) treatment, when the mutant mice had developed the “intestinal obstruction” phenotype. CT images were taken 2, 4, 6, 12, and 24 hours after contrast agent application. Control mice showed completed excretion of the contrast agent within 12 hours after application (D). Intestinal transit in mutant mice was severely impaired, with the contrast agent still detectable in the massively dilated cecum and colon after 12 and 24 hours (I and J).
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
SRF deficiency in adult SM results in impaired contractile activity. Impaired CCh-induced contraction in mutant longitudinal SM. Original recordings from jejunum (A and B), colon (E and F), and urinary bladder (I and J) of 11- to 12-week-old tamoxifen-treated Srfflex1/wt:SMCreERT2/wt (control) and Srfflex1/flex1:SMCreERT2/wt (mutant) mice are shown. The timing is set to CCh addition. Lines indicate 10-μmol/L CCh and 100-μmol/L 3-Isobutyl-1-methylxanthine (IBMX). Amplitudes of the phasic (C, G, and K) and the tonic (D, H, and L) components of contractions were significantly reduced. Bars represent means ± SEMs (n = 15–19 as indicated in the bars). **P < .01, ***P < .001, Student t test.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Analysis of primary colon SMCs. (A) Structural changes of mutant colon SMCs. SMCs of the colon of mutant (Srfflex1/flex1:SM-CreERT2/wt) and control (Srfflex1/wt:SM-CreERT2/wt) mice were isolated 6 days after the last tamoxifen injection and investigated after 7 days of culturing. Control colon SMCs showed typical spindle-shaped structure, ie, elongated cells with distinct cell protrusions (arrows). Mutant colon SMCs lost this SM-specific shape, displaying a protrusion-free flattened and rounded appearance (scale bars, 100 μm). (B) Genotyping of isolated colon SMCs by PCR, showing nearly complete deletion of the Srfflex1 locus in mutant and heterozygous control colon SMCs. DNA of control (Srfflex1/wt:SM-CreERT2/wt; co) and mutant (Srfflex1/flex1:SM-CreERT2/wt; mu) colon SMCs was isolated 7 days after SMC preparation and amplified for genotyping; + indicates positive control. (C) Quantitation of full-length SRF protein levels by Western blotting. Total protein extracts were prepared from the same control (co) and mutant (mu) colon SMCs as in panel B. Total protein (35 μg) was loaded. Reduction of SRF protein level was observed in mutant colon SMCs. (D) Trypan blue exclusion assay of cell viability of colon SMCs. Primary colon SMCs of mutant and control (Srfflex1/wt:SM-CreERT2/wt) mice were isolated 6 days after the last tamoxifen injection. Control and mutant colon SMCs of 3 different mutant mice (Srflx/lx, mouse no. 1–3) were analyzed 12 days after preparation. Mutant colon SMCs showed the same viability compared with control colon SMCs. Results are the mean of 3 different experiments. Error bars correspond to standard deviation (±SD). (E) SAβG staining for senescence of colon SMCs. Control (n = 3) and mutant (n = 3; Srflx/lx, mouse no. 1–3) colon SMCs were analyzed 2 weeks after preparation. Mutant colon SMCs showed high numbers of SAβG-stained cells (asterisks) (scale bars, 50 μm). Quantification of β-galactosidase staining is shown in panel F. Statistic: 3 independent experiments ± SD; ***P < .001, Student t test.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Altered cytoskeletal organization in mutant colon SMCs. (A) Representative control and mutant colon SMCs stained with Phalloidin (red), anti-SRF (green), and DAPI (blue). Mutant colon SMCs showed severely degenerated stress fibers with absent nuclear SRF staining. Cells were fixed and stained 5 days after preparation. Cortical actin fibers are emphasized (white arrows) (scale bars, 100 μm). (B) Real-time PCR showed significantly reduced SM α-actin mRNA levels in mutant colon SMCs (Srflx/lx; mouse no. 1–3). RNA was isolated from cells cultured for 7 days. Each column represents the mean of 3 independent RNA preparations. Error bars correspond to standard deviation. ***P < .001, Student t test. (C) Anti-SM α-actin Western blot of control (co) and mutant (mu) colon SMC protein (30 μg). A weaker SM α-actin signal was observed in mutant compared with control colon SMCs.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Altered expression of SM-specific SRF target genes in primary mutant colon SMCs. Real-time PCR analysis of SM-specific SRF target genes reveals significantly decreased RNA levels of SM22α (A), SM-MHC (B), and smoothelin-A (C) in mutant colon SMCs (Srflx/lx; mouse no. 1–3) compared with control SMCs. RNA was isolated from cells cultured for 7 days. Each column represents the mean of 3 independent RNA preparations and cDNA syntheses, followed by PCR. Error bars correspond to standard deviation. **P < .01; *P < .05, Student t test.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions