Microarray analysis of gene-expression profile in hepatocellular carcinoma cell, BEL-7402, with stable suppression of hLRH-1 via a DNA vector-based RNA interference.

To establish a cell line with a permanent suppression of hLRH-1 in this study, a stable RNAi vector (pSineohLRH-1) targeting hLRH-1 was constructed and introduced into hepatocellular carcinoma cell, BEL-7402. By semiquantitative RT-PCR analysis, the expression of hLRH-1 in BEL-7402 cells carrying pSineohLRH-1 was shown to be significantly suppressed by up to approximately 60%. In addition, microarray analysis was carried out to assess the extent of altered gene expression in BEL-7402 cells with stable knockdown of hLRH-1. Direct comparison of gene-expression profiles of more than 18,000 genes showed that 405 of the expressed genes in hLRH-1-knockdown cells differed dramatically in expression levels from those in controls, which suggested the even extensive biological functions of hLRH-1. Interestingly, among those differentially expressed genes, some are cancer-associated such as Gadd45beta and PTEN, and their expressions were further validated. Although the identification of the exact relationship between these genes and hLRH-1 awaits intensive investigation, the findings of this study provide new insights into the mechanism by which hLRH-1 is involved in tumorigenesis.

Experimental study on the suppression of human nuclear receptor hLRH-1 via a vector-based RNA interference.

To explore the inhibitions of human nuclear receptor hLRH-1 via RNA interference, siRNAs expressing vectors pShLRH-1.1 and pShLRH-1.2, and targeting hLRH-1 were designed and constructed. The recombinants were introduced into hepatocellular carcinoma cells, BEL-7402, mediated by lipofectamin. RT-PCR was carried out to examine the inhibition ratio of hLRH-1 expression. The same method was also applied to analyze the expression of farnesyl pyrophosphate synthetase (FPPS) gene. Our results demonstrated that after transient transfection, both pShLRH-1.1 and pShLRH-1.2 could trigger the efficient inhibition of hLRH-1 in cultured cells, BEL-7402. The inhibition ratios were up to 80%. By comparing with non-transfection and vector-transfection control, the expression of FPPS in cells with inhibition of hLRH-1 was up-regulated significantly. Thus, the inhibition of expression of hLRH-1 in cultured cells was achieved via RNA interference in this study. Our results also suggested that hLRH-1 acts as a negative regulator in FPPS expression.

Generation of transgenic mice with liver-specific expression of human nuclear receptor nr5a2.

Human nuclear receptor hb1 f(nr5a2) was cloned and characterized as a novel member of the Ftz-F1 (nr5a) nuclear receptor subfamily,whose its biological function remained largely unidentified. The aim of this study was to establish transgenic mouse model that specifically expressed hB1F in the liver to faciliate the functional study of hB1F. hb1f cDNA was placed downstream of mouse albumin gene enhancer/promoter to construct a liver-specific hb1f expression vector. Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. Four offspring were identified as carrying the transgenes by PCR,from which one was also verified by Southern blotting. RT-PCR and Western blotting results showed that the transgene was expressed in the liver of the transgenic mice. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 mice were identified by PCR analysis. Genetic analysis of the transgenic mice demonstrated that the transgene had been integrated into the chromosome at a single site and could be stably transmitted.

[Progress in nuclear receptor research].

Nuclear receptors belong to a superfamily of ligand-activated transcription factors, which are involved in regulating gene expression in development, cell differentiation, physiological and metabolic processes. In this review we summarize the studies of nuclear receptor and present the progresses in the researches on nuclear receptor and lipid physiology, nuclear receptor and tumor, and nuclear receptor and co-regulators.

Gene expression profile in liver of hB1F transgenic mice.

AIM: To analyze the tissue morphologic phenotype and liver gene expression profile of hB1F transgenic mice. METHODS: Transgene expression was analyzed with RT-PCR and Western blotting. For one of the transgenic mouse lines, tissue expression pattern of the transgene was also examined with immunochemical methods. Pathological analysis was used to examine the tissue morphologic phenotype of established transgenic mice. The liver gene expression profile of transgenic mice was analyzed with microchip, and some of the differentially expressed genes were verified with RT-PCR. RESULTS: The expressions of hB1F were shown in livers from 6 of 7 transgenic mouse lines. The overexpression of hB1F transgene did not cause pathological changes. Expressions of three genes were up-regulated, while down-regulation was observed for 25 genes. CONCLUSION: The overexpression of hB1F transgene may cause changes of gene expression profiles in the liver of transgenic mice.

Establishment of transgenic mice carrying gene encoding human zinc finger protein 191.

AIM: Human zinc finger protein 191 (ZNF191) was cloned and characterized as a Kruppel-like transcription factor, which might be relevant to many diseases such as liver cancer, neuropsychiatric and cardiovascular diseases. Although progress has been made recently, the biological function of ZNF191 remains largely unidentified. The aim of this study was to establish a ZNF 191 transgenic mouse model, which would promote the functional study of ZNF191. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudo-pregnant female mice. The offsprings were identified by PCR and Southern blot analysis. ZNF 191 gene expression was analyzed by RT-PCR. Transgenic founder mice were used to establish transgenic mouse lineages. The first generation (F1) and the second generation (F2) mice were identified by PCR analysis. Ten-week transgenic mice were used for pathological examination. RESULTS: Four mice were identified as carrying copies of ZNF191 gene. The results of RT-PCR showed that ZNF 191 gene was expressed in the liver, testis and brain in one of the transgenic mouse lineages. Genetic analysis of transgenic mice demonstrated that ZNF 191 gene was integrated into the chromosome at a single site and could be transmitted stably. Pathological analysis showed that the expression of ZNF 191 did not cause obvious pathological changes in multiple tissues of transgenic mice. CONCLUSION: ZNF 191 transgenic mouse model would facilitate the investigation of biological functions of ZNF191 in vivo.

Establishment of transgenic mice carrying the gene of human nuclear receptor NR5A2 (hB1F).

AIM: Human hepatitis B virus enhancer II B1 binding factor (hB1F) was cloned and characterized as a novel member of the Ftz-F1 (NR5A) nuclear receptor subfamily. Although progresses have recently been made, its biological function remains largely unidentified. The aim of this study was to establish an hB1F transgenic mouse model to promote the functional study of hB1F. METHODS: Transgene fragments were microinjected into fertilized eggs of mice. The manipulated embryos were transferred into the oviducts of pseudopregnant female mice. The offsprings were identified by PCR and Southern blot analysis. Transgene expression was analyzed with RT-PCR and Western blot analysis. Transgenic founder mice were used to establish transgenic mouse lineages. The F1 and F2 mice were identified by PCR analysis. RESULTS: Seven mice were identified as carrying copies of transgene. RT-PCR and Western blotting results showed that the transgene was expressed in heart, liver, lung, kidney and stomach in one of the transgenic mouse lineages. Genetic analysis of the transgenic mice demonstrated that the transgene was integrated into the chromosome at a single site, and was transmitted stably. CONCLUSION: In this study we established an hB1F transgenic mouse model, which will facilitate the investigation of the biological function of hB1F in vivo.

[Cloning, genomic organization and promoter activity of the mouse zinc finger protein gene ZF-12].

The human zinc finger protein ZNF191 is a krüppel-like transcription factor, which may be relevant to many diseases such as neuropsychiatric, cardiovascular and liver caner diseases. To elucidate the function of ZNF191 by gene targeting, it is necessary to clone and characterize of the homologous gene in model organisms (mice). The mouse homologous gene (ZF-12) was cloned and sequenced for the first time, the GenBank accession number is AY052495. It contains four exons and three introns; all intronic splice sites exhibited consensus GT/AG sequences. The single nucleotide polymorphisms (SNPs) in exon 2 and the alternative length of 3'-untranslated region (3'-UTR) have been found. The linkage of the ZF-12 gene and the zinc finger protein gene Zfp-35 has been found, so the ZF-12 gene can be localized to B3 to C or beside of chromosome 18. We assessed approximately 1.2 kb of 5'-flanking region of the ZF-12 gene for basal promoter activity. A series of deletion mutants of 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in COS-7 cells, NIH3T3 cells and HeLa cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found that regulatory elements sufficient for basal expression lie between -762 and +70 bp relative to the transcription start site and that a negative regulatory region lie between -824 and -762 bp. This research provides a basis for further study on ZF-12 by gene targeting.

[A new strategy for detecting Lac I mutants based on pMCLac I/neo transgenic mice].

We studied a novel mutation detection method of the two Lac I target genes in pMCLac I/neo transgenic mice. The transgenic mice that contain two types of Lac I genes in pMCLac I/neo vector are different from the transgenic mice carrying only one target gene. Therefore a novel method to detect mutation quickly and efficiently has become a new project after the establishment of pMCLac I/neo transgenic mice. In this paper, a positive selection system--M9/L is used. The result showed that M9/L positive selection system was able to detect the two Lac I target genes, suggesting that it is a rapid and effective method to detect the target mutations in the pMCLac I/neo transgenic mouse.

[Establishment of a hemophilia B transgenic mouse model on the basis of coagulation factor IX gene knock-out mouse].

This study aimed to introduce a site specific point mutation into the human coagulation factor IX gene expressing vectors (pMe4bAIXml plasmid) for microinjection and to obtain transgenic mouse containing copies of a stably integrated pMe4bAIXml plasmid on the basis of coagulation factor IX gene knock-out mouse model as an more efficient animal model of hemophilia b. The site specific point mutation was introduced into pMe4bAIXml plasmid which consists of human coagulation factor IX gene including the entire coding sequence and a shortened first intron, four copies of the mouse MCK enhancer, chicken beta-actin promotor and poly A signal sequence. The vector was linearized and injected into 817 fertilized eggs of mice in which coagulation factor IX gene has been knocked out. The manipulated embryos were transferred into the oviducts of 45 pseudopregnant females, from which 63 offsprings were obtained. The genomic DNAs of these offsprings were analyzed with PCR and genomic Southern blotting. Six mice were found to carry copies of the intact pMe4bAIXml plasmid containing a point mutation and used as founders to establish transgenic mouse lineages.

[p16INK4a exon 1 alpha knockout in mouse embryonic stem cells].

INK4a/ARF locus distinguishes itself by its unusual structure and function. It contains 2 overlapping genes with exons 1 alpha, 2 and 3 encoding p16INK4a and exons 1 beta, 2 and 3 encoding p19ARF. Mice with their exons 2 and 3 of the INK4a/ARF knocked out are viable and fertile but develop spontaneous tumors at an early age and highly sensitive to carcinogenic treatment. However, mice with their exon 1 beta knocked out, without interference the expression of p16INK4a, show almost the same phenotype as those with their exons 2 and 3 knocked out. This raises a question of whether the mouse p16INK4a plays a role in tumor suppression. To investigate this problem, a targeting vector pointing to p16INK4a exon 1 alpha with 1.5 kb Eco81 I/Acc II fragment as short arm and 5.9 kb Xba I/Xho I fragment as long arm was built. After linearlization and purification, the targeting vector was introduced into ES cells through electroporation. Thirty-seven G418- and gancyclovir-resistant colonies were picked out and one of them was confirmed as positive by Southern hybridization.

[Cloning and identification of a mouse zinc finger protein gene ZF-12-related pseudogene].

The mouse zinc finger protein ZF-12 gene is homologous to human gene and encodes a protein of 368 amino acids, which contains four tandem C2H2-type zinc finger motifs in the N-terminal and one SCAN domain in the C-terminal. Some recent studies suggest that ZNF191 might be a hepatocarcinogenesis-associated gene. We screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatis analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any psedogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12.

[Establishment of mouse ZF-12+/- embryonic stem cells].

By using the mouse zinc finger protein gene ZF-12 genomic DNA fragment, pSSC-TV-10.5 was designed and constructed as a replacement vector. Structure of pSSC-TV-10.5 was identified by restrictive digestion analysis and partly sequencing. Then linearized vector was electroporated into ES cells, and transfected cells were screened by G418 and GANC selection. Among 508 G418r/GANCr colonies, 4 were proved to have taken place the homologous recombination of ZF-12 by PCR and southern blotting analysis. This study lays the foundations of preparing mouse models of ZF-12+/- or ZF-12-/-.

Production of Anti-P16 Sera by Genetic Immunization and Protein Immunization.

Genetic immmunization is a new method of producing antibody, which is different from protein immunization. In order to produce anti-P16 sera and to find out the difference between genetic immunization and protein immunization, fusion protein GST-P16 and eukaryotic expression vector pCMV-p16 were injected in rabbit respectively. Western blot analysis showed that the titer of sera from protein immunization was 1:625, which was much higher than the titer from genetic immunization sera. Our results indicate that much has to be done on the characteristics of the genetic immunization and methods for enhancing its immunization effect, before we can use it widely to produce antibody.

Expression of Wild-type and Mutant p16 in H460 Cell Line.

Two p16 mutants, P48L and D74N, were obtained by site-directed mutagenesis using two step PCR method. Mutant p16 cDNA and wild-type p16 cDNA were colned into the mammalian expression vector pcDNA3 to construct p16 expression vector pCMV-p16, pCMV-p16P48L and pCMV-p16D74N, respectively. After the introduction of these expression vectors into human lung cancer cell line H460 in which endogenous p16 gene was homozygously deleted, exogenous p16 expression was detected in G418 resistant cells by Northern blotting and immunocytochemistry staining. The results of immunofluorescence and immunocytochemistry staining showed that the P16 protein was located in the cell cytoplasm. The p16 cDNAs amplificated from the genomic DNA of recombinant H460 cell lines indicated that the plasmid p16 cDNA was integrated into the chromosome of cell lines. That the over expression of wild-type p16 caused G1 arrest suggested the wild-type P16 protein expressed in H460 cell line to be a functional protein.