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The human ZNF268 gene is a novel C2H2 zinc finger gene restrictively expressed in the liver of human embryo. As an initial study on the regulation of its expression, 2.5 kb fragment of the ZNF268 5'-flanking region was cloned from human genome by PCR. This fragment was inserted into pEGFP1 vector and then the recombinant plasmid was transfected into several cell lines by liposomal transfection method. EGFP expression was observed in four cell lines under confocal laser scanning microscope. A series of 5'-deletion fragments from -2456(-)+77 bp to -20(-)+77 bp were inserted into pCAT-Basic vector to construct recombinant reporter plasmids, which were then transfected into Hela cells for deletion analysis. Results of deletion analysis revealed that an important region for transcriptional activity lies between -156 and -20 bp.

The availability of high quality cDNA libraries has proven essential to positional gene cloning efforts differential gene expression studies and EST sequencing/mapping projects.In order to isolate and identify new genes expressed during early human development a 3-week-old human embryo cDNA library was constructed and a pre-screening procedure was used to select cDNAs corresponding to low abundance mRNAs. 6 508 clones were hybridized with a mixture of cDNA probes. Approximately 1 677 clones (26%) did not hybridize with the cDNA probes and represent low abundance mRNAs as well as empty vectors.Partial sequences were generated from one or both end of 47 low abundance cDNA clones and the sequences comparisons with genetic databases revealed that 38.3% of them was annotated human genes 10.6% was highly similar to those from either human or other species 40.4% was partial sequence matched with ESTs that had already been detected and 8.5% of the cDNAs appeared to be unknown in the genetic databases.

The construction, evaluation, and application of cDNA libraries from 3-, 4-, and 5-week-old human embryos are described. Total RNAs were extracted from whole embryos using a modified single-step method. mRNA purified by two passes through oligo (dT) columns was reverse-transcripted into single-stranded cDNA. Alkaline agarose electrophoresis showed that the double-strand cDNA fragments ranged from 0.4 9.0 kb and most of them were in the range of 1.0 2.0 kb. After separation on SizeSep 400 Spun columns to eliminate excess adaptors and small cDNA fragments(less than 400 bp), the cDNAs were ligated into pSPORT1 plasmid and lambdaZipLox phage. The plasmid libraries have complexities of 2.6x10(5), 1.7x10(5) and 2.1x10(5) clones and the phage cDNA libraries have complexities of 3.4x10(6), 3.7x10(6) and 2.3x10(6) clones, respectively. Three whole length cDNAs encoding human CD59, MCP and DAF were amplified by PCR using 3-week-old phage library as templates, and human tPA gene with whole length cDNA was screened from 4-week-old plasmid library by hybridization. It was shown that these libraries are of high quality and are suitable to screen rarely expressed genes. The libraries are a valuable source for the study of novel gene expression during human development.