Figure 1.

Generation of TIMP-3–null animals. (a) Targeting strategy: Approximately 700 bp of Timp-3 promoter sequence and 6 kb from exons 2 and 3 (including the second intron) were placed on either side of a Neo^R cassette in the opposite transcriptional orientation. (b) Southern blot analysis: A flanking Timp-3 or Neo^R cDNA probe detected the mutant allele (18 kb) in heterozygote ES cells and heterozygote and null animals. (c) Northern blot analysis: Abundance of the Timp-3 mRNA was reduced in heterozygote and null animals while expression of a Neo^R mRNA (evidently under the tissue-specific direction of the TIMP-3 promoter) was detected in heterozygote and null animals. (d) Reverse zymographic analysis: TIMP-3 protein was reduced in heterozygotic kidney extracts by approximately half compared with wild-type and was absent in the null extracts.