The Ssh Isolation And Identification Of The Rat Kidney Developmental Genes, Pea3 Gene Expression In Rat Kidney Development And Significance

Part IConstruction and Confirmation of Suppression SubtractiveHybridization Library of Rat Kidney DevelopmentObjective: There is overwhelming information regarding kidney development at the early embryonic stage. However, the later stage of nephrogenic process after birth is poorly understand. We performed suppression subtractive hybridization (SSH) to compare gene expressions in the rat kidney between the two stages of postnatal development: newborn and postnatal 3-week old. Methods: PolyA+ RNA was isolated from newborn rat kidneys and 3-wk-old rat kidneys, respectively. Then double-strand cDNA were synthesized and restricted by Rsa I. Newborn rat kidneys cDNA were divided into two groups and ligated with either adaptor 1 or andptor 2R. After newborn rat kidneys cDNA hybridizated with 3-wk-old rat kidneys cDNA twice and underwent nested PCR, The PCR products were cloned into pGEM(?)-T Easy vector and transformed E.coli XL2-Blue MRF' Ultracompetent Cells. The stated above procedures were also applied to 3-wk-old rat kidneys. Some positive clones randomly picked up were digested and seguenced. Then verified some differentially expressed gene by RT-PCR detection. Results: The SSH library of newborn rat kidneys contained 217 sequences, among which 8 clones are previously unknown seguences, also, 7 represented unknown genes among 142 sequences of the 3-wk-old rat kidneys SSH library. Others derived from 163 known genes can be divided into many categories on the basis of the functions, signal transduction-associated genes, cell cycle, proliferation, gene replication and expression-associated genes, cytoskelet on-associated genes, apoptosis-associated genes, proto...