Gapdh

Heart

Skeletal muscle

Heart

Fig. 5. (A) Southern blot of mouse tail DNA. Mouse tail DNA was digested with April and Pst\ and hybridized with the transgene-specific probe that corresponds to the first chicken /S-actin intron generated by Apal-Xbal digestion of the transgene. The (—) lane represents DNA from transgenenegative littermates and the (+) lane contains DNA from a aB-crystallin transgene-positive mouse. The right lane contains an Apal-Pstl digest of the plasmid containing the aB-crystallin transgene. (B) Northern blot analysis. Total RNA was isolated from mouse heart ventricles and skeletal muscle, and hybridized to 32P-labeled probes corresponding to aB-crystallin. GAPDH was used to demonstrate loading levels. In transgene-positive animals, significant increases in the amount of aB-crystallin occurred in both skeletal muscle and heart. (C) Western blot analysis. A total of 100 /¿g of protein from homogenates of mouse ventricle and skeletal muscle was probed with antibodies against actin and aB-crystallin. In lane 1, 150 ng of recombinant aB-crystallin was loaded as a positive control. A significant increase in the amount of aB-crystallin was found in the heart and skeletal muscle of transgene-positive mice.

Actin aBcrys-tallln

Fig. 5. (A) Southern blot of mouse tail DNA. Mouse tail DNA was digested with April and Pst\ and hybridized with the transgene-specific probe that corresponds to the first chicken /S-actin intron generated by Apal-Xbal digestion of the transgene. The (—) lane represents DNA from transgenenegative littermates and the (+) lane contains DNA from a aB-crystallin transgene-positive mouse. The right lane contains an Apal-Pstl digest of the plasmid containing the aB-crystallin transgene. (B) Northern blot analysis. Total RNA was isolated from mouse heart ventricles and skeletal muscle, and hybridized to 32P-labeled probes corresponding to aB-crystallin. GAPDH was used to demonstrate loading levels. In transgene-positive animals, significant increases in the amount of aB-crystallin occurred in both skeletal muscle and heart. (C) Western blot analysis. A total of 100 /¿g of protein from homogenates of mouse ventricle and skeletal muscle was probed with antibodies against actin and aB-crystallin. In lane 1, 150 ng of recombinant aB-crystallin was loaded as a positive control. A significant increase in the amount of aB-crystallin was found in the heart and skeletal muscle of transgene-positive mice.

as the liver, kidney, and spleen, no aB-crystallin-derived transgene expression can be identified, with the exception of brain, where significant increases in transgene expression also occur.

HSP 70i transgene-positive and -negative littermates are screened by Southern analysis of genomic DNA obtained from tail clips, and their hearts are further analyzed by Northern and Western blotting. Proteins harvested from transgenic mouse heart is probed in a Western blot with a polyclonal antibody that recognizes both constitutive and inducible forms of HSP 70 and with a monoclonal antibody (C92F3A-5, StressGen) that recognizes only the inducible form of HSP 70. As shown in Fig. 6A, the hearts of transgene-positive mice have appreciable HSP 70i immunoreactivity, and the amount of constitutive HSP 70 (HSP 70c) protein does not appear to be altered by the expression of transgene. Figure 6B demonstrates the Northern blot of cardiac and skeletal muscle RNA from a transgene-positive mouse, a transgene-negative mouse, and a transgene-negative mouse subjected to whole body heat stress (8 hr after 42°, 15 min).26 The chimeric transgene (containing the rHSP 70i B form) is transcribed into an mRNA of a unique size due to the addition of sequences derived from the chicken /¡-actin promoter upstream of the translation start site and from SV40 after the translation stop site. The resulting transcript has a size of 2.6 kb and migrates between the mRNAs for the two endogenous mouse HSP 70i transcripts with sizes of 2.7 kb (mHSP 70i A) and 2.5 kb (mHSP 70i B). The novel chimeric rHSP 70i RNA is the transcript responsible for the excess HSP 70i immunoreactivity seen in Fig. 6A. Similar to aB-crystallin transgenic mice, the expression of rHSP 70i transgene is predominantly at heart and skeletal muscle, and some regions of brain.

Using Transgenic Mice to Study Myocardial Ischemia-Reperfusion Injury

The most common method for studying ischemia-reperfusion injury, using transgenic mice, involves an isolated perfused mouse heart preparation. Isolated hearts can be studied in the nonworking Langendorff mode or in working mode. The working mouse heart preparation is difficult and requires sophisticated techniques and skills. Whereas working mouse heart preparations are used in only a handful of laboratories, nonworking mouse hearts are extensively used.

Isolated Nonworking Mouse Heart Preparation and Measurement of Contractile Function

Mice are anesthetized and the hearts are excised after thoracotomy. The aorta is cannulated, and the heart is perfused with Krebs-Henseleit bicarbonate (KHB)

26 R. Hotchkiss, I. Nunnally, S. Lindquist, J. Taulien, G. Perdrizet, and G. Karl, Am. J. Physiol. 265, R1447 (1993).

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