1. Volume 2, Issue 5, Pages 559-569 (November 1998)
A Muscle-Specific Insulin Receptor Knockout Exhibits Features of the Metabolic Syndrome of NIDDM without Altering Glucose Tolerance 
Jens C Brüning, M.Dodson Michael, Jonathon N Winnay, Tatsuya Hayashi, Dieter Hörsch, Domenico Accili, Laurie J Goodyear, C.Ronald Kahn 
Molecular Cell 
Volume 2, Issue 5, Pages (November 1998)
DOI: /S (00)

2. Figure 1 LoxP Targeting of the IR Gene
(a) The upper diagram shows the targeting construct to introduce loxP sites into the IR gene. The middle diagram shows the simplified restriction map of the murine IR gene surrounding exon 4 (shaded box), and the lower diagram indicates the IR gene after homologous recombination. Probe 1 indicates the position of the DNA fragment outside the region of homology in intron 3 of the IR gene used to identify homologous recombinant (HR) clones.
(b) Southern blot analysis with probe 1 of different ES cell clones after transfection with the targeting construct. Clone #35 shows the expected bands of 14 kb and 9.5 kb for a cell line in which one copy of the endogenous IR gene was replaced by the targeting construct (IR(lox) allele).
(c) Schematic representation of the IR(lox) allele after transient expression of Cre recombinase with subsequent deletion of the selection cassette. P1 and P2 indicate the position of the primers used for the PCR analysis to detect the loxP site, and probe 2 shows the probe corresponding to the neo coding sequence. The primers P1 and P2 were used for PCR-based genotyping of the IR(lox) locus.
(d) The left panel shows Southern blot analysis of DNA extracted from a homologous recombinant clone before transient Cre expression (#35) and ganciclovir-resistant subclones of this clone after Cre transfection (35.4, 35.7, 35.1, 35.8) using probe 2. The right panel shows a PCR analysis of the same clones, indicating that subclones 35.1 and 35.8 have lost the resistance cassette but retained the loxP site downstream of exon 4.
Molecular Cell 1998 2, DOI: ( /S (00) )

3. Figure 2 Muscle-Specific Expression of Cre Recombinase
(a) Schematic of the muscle creatine kinase promoter-Cre cDNA-SV40 poly(A) transgene. Demonstrated is the structure of the 5′ regulatory region of the Mck gene encompassing a 3.3 kb promoter fragment, the untranslated exon 1, a 3 kb intron 1 with enhancer 2. pA, and the SV40 polyadenylation signal.
(b) The left panel shows the Northern blot analysis of total RNA isolated from muscle of a wild-type mouse (WT) or offspring from different MCK-Cre founder mice (2–6). The blot was probed with a 32P-labeled, full-length Cre cDNA. The arrow indicates the presence of the predicted size of the mature message. The right panel shows Northern blot analysis of total RNA extracted from different tissues from an offspring of founder MCK–Cre 5. The blot was probed with the same probe used in the left panel.
Molecular Cell 1998 2, DOI: ( /S (00) )

4. Figure 3
MIRKO Mice Exhibit High-Efficiency Inactivation of the IR Gene Selectively in Skeletal Muscle and Heart
(a) Immunoprecipitation and Western blot analysis on hind limb muscle extracts (5 mg) from WT (open bar), IR(lox/lox) (hatched bar), and MIRKO (closed bar) mice using an IR-specific antiserum. The graph shows the quantitation of IR content as percent of IR content in muscle from WT mice.
(b) Protein extracts (5 mg) from different muscles of WT, IR(lox/lox), and MIRKO mice were subjected to immunoprecipitation with an IR-specific antiserum followed by Western blot analysis using the same antibody. Quantification of the level of IR protein in duplicate samples from three individual animals of each genotype was performed by phosphorimager analysis as described in Experimental Procedures. Muscles from six individual animals of each genotype were pooled in order to have sufficient protein for analysis of IR levels in soleus and EDL. A representative blot is shown for each tissue. Quad, quadriceps; Gastroc, gastrocnemius; Glut, gluteus; Sol, soleus; EDL, extensor digitorum longus; Tric, triceps; Pect, pectoralis.
(c) Immunohistochemical analysis of muscle isolated from WT (upper panel), IR(lox/lox) (middle panel), and MIRKO (lower panel) mice using an IR-specific antiserum. Note the lack of IR expression in muscle fibers from MIRKO mice (m), whereas contaminating adipose tissue (a) exhibits staining equal to WT and IR(lox/lox) mice. Controls using no primary antiserum or nonimmune antiserum did not exhibit staining in adjacent sections (data not shown).
(d) Protein extracts (fat, 2.5 mg; other tissues, 5 mg) were prepared from tissues of WT, IR(lox/lox), and MIRKO mice (three per genotype) and subjected to immunoprecipitation with an IR-specific antiserum followed by Western blot analysis using the same antibody. Quantification of the level of IR protein by phosphorimager analysis revealed a range of 83%–120% of the WT IR expression level in IR(lox/lox) and MIRKO tissues (p = ns). A representative blot is shown for each tissue.
Molecular Cell 1998 2, DOI: ( /S (00) )

5. Figure 4
Insulin-Stimulated Signaling Events in Liver and Skeletal Muscle from WT, IR(lox/lox), and MIRKO Mice
(a) WT, IR(lox/lox), and MIRKO mice were anaesthetized by intraperitoneal injection of pentobarbital and injected with either saline (−) or 5 IU of regular insulin (+) via the inferior vena cava. The liver was removed after 1 min, and protein extracts were subjected to immunoprecipitation with an IR-specific antiserum followed by Western blot analysis with an anti-phosphotyrosine-specific antibody (upper panel) or an IR-specific antiserum (lower panel).
(b) The experiment was performed as described in (a) with the exception that skeletal muscle was removed 3 min after saline or insulin injection.
(c) The experiment was performed as described in (a) with the exception that immunoprecipitations were performed with an IRS-1-specific antiserum followed by Western blot analysis with an anti-phosphotyrosine-spe-cific antibody (upper panel), an IRS-1-specific antiserum (second panel from top), or an antiserum to the p85 regulatory subunit of PI 3-kinase (second panel from bottom). The bottom panel shows a Western blot analysis on the same extracts using an anti-p85 antiserum.
(d) The experiment was performed as described in (c) with the exception that proteins were extracted from skeletal muscle isolated 3 min after saline or insulin injection.
Molecular Cell 1998 2, DOI: ( /S (00) )

6. Figure 5
Insulin-Stimulated Glucose Transport in Isolated Skeletal Muscle from WT, IR(lox/lox), and MIRKO Mice
(a) Female mice were sacrificed by cervical dislocation, and glucose transport was determined on isolated soleus muscle as described under Experimental Procedures. Muscles from WT, IR(lox/lox), and MIRKO mice were untreated (open bars) or stimulated with 6.6 nM (hatched bars) or 33 nM (closed bars) human regular insulin.
(b) Experiments were performed as described for (a) with the exception that glucose transport was determined in EDL muscle from WT, IR(lox/lox), and MIRKO mice.
Molecular Cell 1998 2, DOI: ( /S (00) )

7. Figure 6
Physiological Consequence of Mus-cle-Specific IR Gene Inactivation
(a) Triglyceride levels were determined on serum samples from overnight-fasted, 4-month-old WT (open bar), IR(lox/lox) (hatched bar) and MIRKO (closed bar) mice. Each bar represents the mean of at least ten animals of each genotype ±SEM. Double asterisk, p < 0.01.
(b) Free fatty acid levels were determined on serum samples from overnight-fasted, 4-month-old WT (open bar), IR(lox/lox) (hatched bar), and MIRKO (closed bar) mice. Each bar represents the mean of at least ten animals of each genotype ±SEM. Asterisk, p < 0.05.
(c) Blood glucose concentrations were determined on venous blood samples from random fed, 4-month-old WT (open bar), IR(lox/lox) (hatched bar), and MIRKO (closed bar) mice. Each bar represents the mean of at least ten animals of each genotype ±SEM.
(d) Insulin concentrations were determined on serum from random fed, 4-month-old WT (open bar), IR(lox/lox) (hatched bar), and MIRKO (closed bar) mice. Each bar represents the mean of at least ten animals of each genotype ±SEM.
(e) Glucose tolerance tests were performed on 4-month-old WT (circle), IR(lox/lox) (triangle), and MIRKO (square) mice as described under Experimental Procedures. Results are expressed as mean blood glucose concentration ±SEM from at least eight animals of each genotype.
(f) Insulin tolerance tests were performed on 4-month-old WT (circle), IR(lox/lox) (triangle), and MIRKO (square) mice as described under Experimental Procedures. Results are expressed as mean percent of basal blood glucose concentration ±SEM from at least eight animals of each genotype.
Molecular Cell 1998 2, DOI: ( /S (00) )

8. Figure 7
Potential Mechanisms Compensating for the Lack of Functional IR Expression in Skeletal Muscle of MIRKO Mice
(a) Protein extracts prepared from skeletal muscle of WT, IR(lox/lox), and MIRKO mice were subjected to Western blot analysis using an anti-IGF-1-receptor-specific antiserum (upper panel) or an anti-Glut4-specific antiserum (middle panel). To test whether insulin induces any tyrosine kinase activity in skeletal muscle of MIRKO mice, animals were either untreated (−) or injected with 5 U of human regular insulin (+), whereafter protein extracts were prepared from skeletal muscle. These extracts were subjected to immunoprecipitation using an anti-phosphotyrosine-specific antibody followed by Western blot analysis using the same antibody (lower panel).
(b) Epididymal fat pads from 4-month-old, male WT (open bar), IR(lox/lox) (hatched bar), and MIRKO (closed bar) mice were dissected and weighed. Results are expressed as the mean weight ±SEM. Asterisk, MIRKO versus WT + IR(lox/lox), p = 0.02.
Molecular Cell 1998 2, DOI: ( /S (00) )

9. Figure 8
Functional IR Expression in Skeletal Muscle Is Required to Maintain Euglycemia in IR(+/−) Mice
(a) Blood glucose concentrations were determined on venous blood samples from random fed, 3-month-old WT (open bar), MIRKO (hatched bar), IR(+/−) (cross-hatched bar), and MIRKO−50% (closed bar) mice. Each bar represents the mean of at least seven animals of each genotype ±SEM.
(b) Plasma insulin concentrations were determined on venous blood samples from random fed, 3-month-old WT (open bar), MIRKO (hatched bar), IR(+/−) (cross-hatched bar), and MIRKO−50% (closed bar) mice. Each bar represents the mean of at least seven animals of each genotype ±SEM. Double asterisk, p < 0.01.
(c) Insulin tolerance tests were performed on 3-month-old WT (circles), IR(+/−) (triangles), and MIRKO-50% (squares) mice as described under Experimental Procedures. Results are expressed as mean percent of basal blood glucose concentration ±SEM from at least seven animals of each genotype.
(d) Blood glucose concentrations were determined from venous blood samples from random fed, 6-to-8-month-old WT, MIRKO, IR(+/−), and MIRKO−50% mice. Data are presented as values determined from individual animals (circle, females; triangle, males).
(e) Plasma insulin concentrations were determined from venous blood samples from random fed, 6-to-8-month-old WT, MIRKO, IR(+/−), and MIRKO−50% mice. Data are presented as values determined from individual animals (circle, females; triangle, males).
Molecular Cell 1998 2, DOI: ( /S (00) )