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The cDNA for the inducible rat heat shock protein 70 gene has been cloned in our laboratory24 and is cleaved from a plasmid by digestion with Xbal and Sacl. To this fragment EcoRl-Xbal and SacI-£coRI converters are ligated, as described above. The EcoRl-Xbal converter has the sequence 5'-AATTCGATCTCGAT-3' on the upper coding strand and is also annealed to a complementary oligonucleotide so that a 5' EcoRI site and a 3' Xbal site are generated. The Sac\-EcoR\ converter has the sequence 5'-CGATCTGCATTGAG-3' on the upper coding strand and is also annealed to a complementary oligonucleotide so that a 5' Sacl site and a 3' EeoRI site are generated. The rHSP 70i cDNA fragment modified in this way can be ligated to the £coRI-digested plasmid pCAGGS as described above and the resulting transgene construct is shown in Fig. 4 (right). This transgene construct is then digested with Sail and Sacl to yield a linear fragment that contains the enhancer-promoter followed by an intron upstream of the rHSP 70i cDNA. On the 3' side of the cDNA a simian virus 40 (SV40) polyadenylation signal is present to confer proper termination and stabilization of the expressed transgene mRNA.

Both transgene fragments are purified from the rest of the vector backbone in a 0.8% (w/v) low melting point agarose gel from which they are subsequently isolated by passing the melted and diluted agarose through an Elutip (Schleicher & Schuell, Keene, NH) column. After dialysis against injection buffer [7.5 mMTris (pH 7.4), 0.15 mM EDTA], 1-2 ¿¿1 of the DNA fragment is injected into the pronuclei of fertilized eggs at a concentration of 2 /xg/ml. In the case of aB-crystallin we have derived two transgenic lines, ccB 1 and aB2, of which the aBl line has the highest transgene expression levels and is used primarily for the reported studies. In the case of rHSP 70i we have several founder lines that overexpress the inducible HSP 70 mRNA and protein at fairly equal levels. Representative Southern, Northern, and Western blots are discussed below.

Characterization ofaB-Crystallin and Heat Shock Protein 70 Transgenic Mice aB-Cry stallin transgenic mice are identified by Southern blot analysis of mouse tail DNA with a transgene-specific probe25 (Fig. 5A). As shown in Fig. 5B, in the hearts of heterozygous aBl mice, aB-crystallin mRNA is 3.1-fold elevated above the level of that in control mice. There is a 6.9-fold increase at the protein level (Fig. 5C). Endogenous aB-crystallin expression in the rodent heart is relatively high. In mouse heart extract, a basal level of «B-cry stall in constitutes close to 0.1' of total protein. The level of transgene expression in the heart is high. In addition, significant transgene expression occurs in skeletal muscle and the increase is even more dramatic than that observed in the heart. A 4.8-fold increase is seen at the mRNA level and a 7.9-fold increase is seen at the protein level. In other organs such

24 R. Mestril, S. H. Chi, M. R. Sayen, and W. H. Dillmann, Biochem. J. 298, 561 (1994).

25 P. S. Ray, J. L. Martin, E. A. Swanson, H. Otani, W. H. Dillmann, and D. K. Das, FASEB J. 15, 393 (2001).

A + pCAGGS

aB Crystallin

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