Cloning of Chromosomal Band Specific cDNA - cDNA related with neural development- / 대한해부학회지

Recently, surmountable amounts of genes are being cloned without information about them and it has become neccessary to develop new techniques for discovering genes with more informaiion like as chromosomal location and possible functions. We have developed one such a method and applied it to search for genes that may be related with the neural development. The mRNAs were extracted from cerebral cortex of 18 week old human fetus, cDNAs were made by reverse transcription from these mRNAs and Uni-amp cDNAs having Uni-amp adapters at both ends were made for subsequent PCR. To observe the distribution of the Uni-amp cDNAs on the chromosome, fluorescent in situ hybridization was performed with biotin labeled Uni-amp cDNAs. Among the chromosome bands showing strong hybridization with the cDNAs, 7q22 was microdissected from the chromosome hybridized with unlabeled Uni-amp cDNAs and amplified by PCR with Uni-amp primers. These amplified cDNA fragment were subcloned to vectors and the nucleic acid sequences were analysed. As a result, 46 different clones were obtained. They were categorized as 12 clones of well characterized genes, 14 clones showing low homology with known genes, 13 clones of simply registered uncharacterized human cDNAs, 7 clones of unknown genes. In situ hybridization histochemistry of 34 novel genes, except 12 known genes, were performed on developing and adult rat tissue sections to see the tissue specificity and developmental expression of these genes. The expression of several novel genes were restricted to the nervous system. From these results, it may be suggested that our technique is very useful to clone the genes expressed in the developing human braine with confirmed chromosomal location. In addition, this cloning technique can be used to discover the new genes related with neural development in combination with functional screening methods.

The Developmental Differences of Damage in Rat Brain by Systemic Kainic Acid Injection / 대한해부학회지

Kainic acid[KA], a glutamic acid analogue, has been widely used as a excitotoxin in the study of neurotoxicity due to its ability to induce strong excitation and to increase intracellular calcium concentration of the mature central nervous system[CNS] neurons. However, it is not well known whether KA is also strongly cytotoxic to the neurons of the postnatal brain. We have injected KA into rats at different developmental stages and have investigated the changes in the expression of c-fos[transcriptional factor and a marker of neuronal activity], heat shock proetin 72[hsp 72, a neuronal injury marker], and glial fibrillary acidic protein[GFAP, a neuronal injury marker] mRNAs, which are known to be increased in KA-induced neurotoxicity, and glyceraldehyde 3-phosphate dehydrogenase [GAPDH, a house keeping gene] mRNAs with in situ hybridization histochemistry using specific riboprobes. The expression of c-fos mRNA was first identified in the CA3 area of hippocampus from 6hr after KA treatment in P7 rats. The c-fos mRNA-expressing area and the level of expression was gradually increased from P7 to adult. Hsp 72 mRNA was first expressed in the dentate gyrus and hippocampus from 6hr after KA treatment in P2l rats. In the adult rats, hsp 72 mRNA was broadly expressed in the brain at 2hr after KA treatment. The increase of GFAP mRNA expression was first identified in Pl4 rat brain from 6hrs after KA treatment, and by the development of brain it tends to appear earlier. The expression of GAPDH mRNA, however, did not show changes after KA treatment except for the adult rats showing a slight decrease at 12hr after KA treatment. These results suggest that KA may offer different level of cytotoxicity to the developing neurons by their developmental status and the difference may be correlated with the completion of synaptogenesis and increase of KA receptor.