Representational difference analysis of cDNA of genes expressed in embryonic kidney

Abstract

Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based differential cloning method. It involves hybridization of two populations of cDNA with selective amplification of differentially expressed genes. To isolate the differentially expressed genes during renal development, mRNAs from embryonic kidneys at day 13 (E13) and postnatal kidneys from three-week-old (P3) mice were extracted, and double stranded cDNAs prepared. Double stranded cDNAs were digested with DpnII, adaptor-ligated, and amplified by PCR, using adaptor primer to generate "representative amplicons." These reflect the "representation" of most of the cDNA population. The term "amplicons" denotes amplified PCR product. Among the two populations of cDNA, E13 kidney cDNA was used as a "tester," containing target genes, and P3 kidney cDNA as a "driver," driving the process of subtraction, following which, they were subjected to cDNA-RDA under low stringency conditions. During the first round of cDNA-RDA embryonic globin genes were isolated. To competitively eliminate these genes, plasmid DNAs of globin genes were supplemented into driver, and subjected to the second round of cDNA-RDA. This resulted in the isolation of four cDNA clones: H19 gene, mesoderm-specific cDNA, COL2A1 gene, and a novel cDNA. By Northern blot analyses, the H19 gene and mesoderm-specific cDNA exhibited a high degree of developmental regulation, that is, they were abundantly expressed in E13 kidney, and their expression was barely detectable in P3 kidney. The differential developmental regulation of mesoderm-specific cDNA was confirmed by tissue in situ hybridization experiments. The COL2A1 and novel cDNA were rare transcripts in the embryonic Kidney. However, Southern blot analyses of representations indicated their up-regulated expressions in E13 kidneys. The novel gene was differentially expressed in 13-day embryonic lung, and Northern blot analysis revealed an approximately 10 Kb transcript. These results indicate that cDNA-RDA is a sensitive technique to identify rare transcripts with differential expression, and since there is a minimal chance to isolate false positive clones, cDNA-RDA may serve as a powerful tool for delineating up- or down-regulation of the genes involved in various pathological or physiological states of the kidney.