MicroRNA-7 inhibits tumor growth and metastasis by targeting the phosphoinositide 3-kinase/Akt pathway in hepatocellular carcinoma.

UNLABELLED: MicroRNAs (miRNAs) are known to be involved in carcinogenesis and tumor progression in hepatocellular carcinoma (HCC). Recently, microRNA-7 (miR-7) has been proven to play a substantial role in glioblastoma and breast cancer, but its functions in the context of HCC remain unknown. Here, we demonstrate that miR-7 inhibits HCC cell growth and metastasis in vitro and in vivo. We first screened and identified a novel miR-7 target, phosphoinositide 3-kinase catalytic subunit delta (PIK3CD). Overexpression of miR-7 would specifically and markedly down-regulate its expression. miR-7-overexpressing subclones showed significant cell growth inhibition by G(0) /G(1) -phase cell-cycle arrest and significant impairment of cell migration in vitro. To identify the mechanisms, we investigated the phosphoinositide 3-kinase (PI3K)/Akt pathway and found that Akt, mammalian target of rapamycin (mTOR), and p70S6K were down-regulated, whereas 4EBP1 was up-regulated in miR-7-overexpressing subclones. We also identified two novel, putative miR-7 target genes, mTOR and p70S6K, which further suggests that miR-7 may be a key regulator of the PI3K/Akt pathway. In xenograft animal experiments, we found that overexpressed miR-7 effectively repressed tumor growth (3.5-fold decrease in mean tumor volume; n = 5) and abolished extrahepatic migration from liver to lung in a nude mouse model of metastasis (n = 5). The number of visible nodules on the lung surface was reduced by 32-fold. A correlation between miR-7 and PIK3CD expression was also confirmed in clinical samples of HCC. CONCLUSION: These findings indicate that miR-7 functions as a tumor suppressor and plays a substantial role in inhibiting the tumorigenesis and reversing the metastasis of HCC through the PI3K/Akt/mTOR-signaling pathway in vitro and in vivo. By targeting PIK3CD, mTOR, and p70S6K, miR-7 efficiently regulates the PI3K/Akt pathway. Given these results, miR-7 may be a potential therapeutic or diagnostic/prognostic target for treating HCC.

Role and fate of SP100 protein in response to Rep-dependent nonviral integration system.

Previously, we studied an AAVS1 site-specific non-viral integration system with a Rep-donor plasmid and a plasmid containing adeno-associated virus integration element. Our earlier study focused on the plasmid vector itself, but the cellular response to the system was still unknown. SP100 is a member of the promyelocytic leukemia nuclear bodies. It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. In this study, we revealed that SP100 inhibited the Rep-dependent nonviral integration. Conversely, transient expression of Rep78 increased the degradation of SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome. SP100 and Rep78 are both located in the nucleolus, which provides the spatial possibility for their interaction. Rep78 was coimmunoprecipitated with the enhanced green fluorescent protein (EGFP)-SP100 fusion protein but not EGFP, which verified the interaction between Rep78 and SP100. These results have enriched our knowledge about the cellular protein SP100 and Rep-dependent nonviral integration. It may lead to an improvement in the application of Rep-related transgene integration method and in the selection of target cells.

The 156KELK159 tetrapeptide of HIV-1 integrase is critical for lentiviral gene integration.

HIV-1 integrase (HIV-1 IN), a key element of HIV-1-derived lentiviral vectors, is crucial for the stable maintenance of the vector gene by inserting them into host genome. HIV-1 IN has been found to have functions other than integration, such as involving in virion morphology, viral DNA synthesis and viral DNA nuclear import. In our study, the yeast two-hybrid assay identified a tetrapeptide 156KELK159 in HIV-1 IN that was crucial for HIV-1 IN and Daxx interaction. To investigate the functions of the tetrapeptide 156KELK159 of the HIV-1 IN, both the wild type HIV-1 IN and a mutant without 156KELK159 were used to package the EGFP reporter gene contained lentivirus. p24 based titer assay revealed that deleting the tetrapeptide did not affect virus packaging. The result was verified by quantitative real time PCR with viral specific primers. But the 156KELK159 was crucial for lentiviral gene integration. Deleting the tetrapeptide made the percentage of cells expressing the reporter gene significantly decreased and did not affect the level of DNA entered into the cells or nucleus. Real time reverse transcription PCR and FACS were used to detect the lentiviral report gene expression in infection maintaining cells and revealed 156KELK159 did not affect lentiviral vector gene expression. Our results may shed light on the regulatory mechanism of gene integration of lentivirus.

Cdc20 mediates D-box-dependent degradation of Sp100.

Cdc20 is a co-activator of the anaphase-promoting complex/cyclosome (APC/C complex), which recruits substrates at particular phases of the cell cycle and mediates their degradation. Sp100 is a PML-NB scaffold protein, which localizes to nuclear particles during interphase and disperses from them during mitosis, participates in viral resistance, transcriptional regulation, and apoptosis. However, its metabolism during the cell cycle has not yet been fully characterized. We found a putative D-box in Sp100 using the Eukaryotic Linear Motif (ELM) predictor database. The putative D-box of Sp100 was verified by mutational analysis. Overexpression of Cdc20 resulted in decreased levels of both endogenous Sp100 protein and overexpressed Sp100 mRNA in HEK 293 cells. Only an overexpressed D-box deletion mutant of Sp100 accumulated in HEK293 cells that also overexpressed Cdc20. Cdc20 knockdown by cdc20 specific siRNA resulted in increased Sp100 protein levels in cells. Furthermore, we discovered that the Cdc20 mediated degradation of Sp100 is diminished by the proteasome inhibitor MG132, which suggests that the ubiquitination pathway is involved in this process. However, unlike the other Cdc20 substrates, which display oscillating protein levels, the level of Sp100 protein remains constant throughout the cell cycle. Additionally, both overexpression and knockdown of endogenous Sp100 had no effect on the cell cycle. Our results suggested that sp100 is a novel substrate of Cdc20 and it is degraded by the ubiquitination pathway. The intact D-box of Sp100 was necessary for this process. These findings expand our knowledge of both Sp100 and Cdc20 as well as their role in ubiquitination.

PhiC31 integrase interacts with TTRAP and inhibits NFkappaB activation.

Phage PhiC31 integrase-mediated gene delivery is believed to be safer than using retroviral vectors since the protein confines its insertion of the target gene to a limited number of sites in mammalian genomes. To evaluate its safety in human cells, it is important to understand the interactions between this integrase and cellular proteins. Here we show that PhiC31 integrase interacts with TTRAP as presented by yeast two-hybrid and co-immunoprecipitation assays. Reducing the expression of endogenous TTRAP can increase the efficiency of PhiC31 integrase-mediated integration. A possible effect of interaction between PhiC31 integrase and TTRAP was highlighted by the fact that PhiC31 integrase inhibited the NFkappaB activation mediated by IL-1 in a dose-dependent manner. Because low dose of PhiC31 integrase can mediate considerable recombination events, we suggest that low dose of PhiC31 integrase be used when this integrase is applied in human cells.

Cellular protein TTRAP interacts with HIV-1 integrase to facilitate viral integration.

TTRAP is a PML-NB protein that is involved in the NF-kappaB signaling pathway. TTRAP was recently identified by yeast two-hybrid analysis as a HIV-1 integrase (HIV-1 IN) interacting protein. This interaction was verified by co-immunoprecipitation, GST pull-down, and intracellular imaging, and deletion assays suggested that the N-terminal 180 residues of TTRAP are responsible for the interaction. In stable TTRAP knock-down cell lines, the integration of viral vectors decreased significantly compared with non-silenced cell lines. Conversely, overexpression of TTRAP by transient transfection increased the percentage of integration events. This is the first time that TTRAP has been shown to interact with HIV-1 IN and facilitate lentiviral vector integration. These findings reveal a new function of TTRAP and expand our understanding of the cellular response to HIV infection. The interaction between TTRAP and HIV-1 IN may be useful in designing new anti-viral strategies as well as for improving the efficiency of lentiviral-vector-mediated gene delivery.

Daxx interacts with HIV-1 integrase and inhibits lentiviral gene expression.

The death-associated protein Daxx is a ubiquitously expressed gene in mammals and is widely involved in transcriptional regulation and cellular intrinsic immune response against incoming virus. We found here that knocking down endogenous Daxx with specific siRNA increased HIV-1-derived lentiviral reporter gene expression in 293T cells. This repressive effect of Daxx is not due to its inhibition on viral gene integration into the cellular genome and is independent of the ubiquitin promoter on the vFUGW lentiviral vector. Instead, this inhibition is dependent on Daxx's interaction with HIV-1 integrase. A histone deacetylases (HDACs) inhibitor increased reporter gene expression to the level similar to Daxx knockdown in vFUGW infected cells but there was no additive effect in combination of HDACs inhibitor and Daxx-specific siRNA. Our results suggest that Daxx may associate with HIV-1-derived lentiviral DNA via interacting with HIV-1 integrase and recruit HDACs to viral DNA to repress lentiviral gene expression.

TTRAP is a novel PML nuclear bodies-associated protein.

PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-gamma, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.

DAXX interacts with phage PhiC31 integrase and inhibits recombination.

Phage PhiC31 integrase has potential as a means of inserting therapeutic genes into specific sites in the human genome. However, the possible interactions between PhiC31 integrase and cellular proteins have never been investigated. Using pLexA-PhiC31 integrase as bait, we screened a pB42AD-human fetal brain cDNA library for potential interacting cellular proteins. Among 61 positives isolated from 10(6) independent clones, 51 contained DAXX C-terminal fragments. The strong interaction between DAXX and PhiC31 was further confirmed by co-immunoprecipitation. Deletion analysis revealed that the fas-binding domain of DAXX is also the region for PhiC31 binding. Hybridization between a PhiC31 integrase peptide array and an HEK293 cell extract revealed that a tetramer, 451RFGK454, in the C-terminus of PhiC31 is responsible for the interaction with DAXX. This tetramer is also necessary for PhiC31 integrase activity as removal of this tetramer resulted in a complete loss of integrase activity. Co-expression of DAXX with PhiC31 integrase in a HEK293-derived PhiC31 integrase activity reporter cell line significantly reduced the PhiC31-mediated recombination rate. Knocking down DAXX with a DAXX-specific duplex RNA resulted in increased recombination efficiency. Therefore, endogenous DAXX may interact with PhiC31 causing a mild inhibition in the integration efficiency. This is the first time that PhiC31 was shown to interact with an important cellular protein and the potential effect of this interaction should be further studied.

Cloning and expression of translation elongation factor 2 (EF-2) in zebrafish.

We have identified a developmentally regulated gene translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame from nucleotide 55 to 2631 and encodes a protein of 858 amino acids. It shares an identity of 92, 93, 93, 92, 79 and 80% in amino acid sequence to human, mouse, Chinese hamster, Gallus gullus, C. elegans and Drosophila EF-2, respectively. Zebrafish EF-2 protein has 16 conserved domains, GTP-binding domain is found in the NH2 terminus, and the ADP-ribosylation domain locates at the COOH terminus. Whole mount in situ hybridization on zebrafish embryos shows that the transcripts of EF-2 gene are detected during the early development of zebrafish embryo and constantly change from 5-somite stage to protruding-mouth stage. It expresses strongly throughout envelope at 5-somite stage. Then the stained cells concentrate strongly in the eyes, brain and muscle tissue. From prim-25 stage the stained cells only appear strongly in the lens and the anterior portion of the cerebellum.

Cloning, expression and functional study of translation elongation factor 2 (EF-2) in zebrafish.

We have identified translation elongation factor 2 (EF-2) in zebrafish (GenBank Accession No. AAQ91234). Analysis of the DNA sequence of zebrafish EF-2 shows that the 2826 bp cDNA spans an open reading frame between nucleotide 55 to 2631 and encodes a protein of 858 amino acids. Zebrafish EF-2 protein shares 92%, 93%, 93% and 92% identity with the corresponding amino acid sequence in human, mouse, Chinese hamster and Gallus EF-2, respectively. Whole-mount in situ hybridization showed that zebrafish EF-2 was a developmentally regulated gene and might play important roles during the early development of zebrafish embryos. Therefore, we further studied the function of EF-2 during early embryogenesis. Using morpholino antisense oligo knockdown assays, anti-MO injected embryos were found to display abnormal development. The yolk balls were larger than normal and the melanophores spreading on their bodies became fewer. Furthermore, their tails were incurvate and their lenses were much smaller than those of the normal embryos. However the EF-2 overexpression data showed that extra EF-2 protein had no obvious effect on zebrafish embryonic development.

Phyllanthus emblica Linn. fruit extract potentiates the anticancer efficacy of mitomycin C and cisplatin and reduces their genotoxicity to normal cells in vitro.

OBJECTIVE: Fruit of Phyllanthus emblica Linn. (PE) is widely consumed as a functional food and used as a folk medicine due to its remarkable nutritional and pharmacological effects. Mitomycin C (MMC) and cisplatin (cDDP) are the most widely used forms of chemotherapeutic drug, but their clinical use is limited by their genotoxicity to normal cells. We aimed to determine whether PE has potential to reduce the genotoxicity, while improving the anticancer effect, of MMC and cDDP. METHODS: Cell proliferation was evaluated using the trypan blue exclusion assay and colony-forming assay. Genomic instability (GIN) was measured using the cytokinesis-block micronucleus assay. RESULTS: Co-treatment (72 h) with PE at 20-320 µg/ml significantly enhanced the efficacy of MMC (0.05 µg/ml) and cDDP (1 µg/ml) against Colo205 colorectal cancer cells (P<0.05), and at 80-320 µg/ml significantly decreased MMC- and cDDP-induced GIN and multinucleation in normal colonic NCM460 cells (P<0.05). PE significantly decreased the mitotic index (P<0.01), blocked mitotic progression (P<0.05), and promoted apoptosis (P<0.01) in MMC- and cDDP-treated NCM460 cells, suggesting that PE-mediated inhibition of mitosis and induction of apoptosis may limit the division and survival of highly damaged cells. Also, PE was found to inhibit the clonal expansion of MMC- and cDDP-treated NCM460 cells (P<0.05) and decrease the heterogeneity of the surviving clones. CONCLUSIONS: PE potentiates the anticancer efficacy of MMC and cDDP, while preventing their genotoxicity and inhibiting clonal expansions of unstable genomes in normal cells. These data suggest that PE has the potential to reduce the risk of secondary cancers induced by chemotherapeutics.

Association between the MTHFR C677T polymorphism, blood folate and vitamin B12 deficiency, and elevated serum total homocysteine in healthy individuals in Yunnan Province, China.

BACKGROUND: An increased serum total homocysteine (tHcy) concentration is typically associated with genetic defects involved in Hcy metabolism or related nutritional deficiencies. In this study, the combined effects of methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism and folate and vitamin B12 deficiency on serum total Hcy (tHcy) levels were evaluated in a healthy Chinese population in Yunnan Province, China. METHODS: The MTHFR C677T polymorphism was genotyped in 330 volunteers (164 men and 166 women) using polymerase chain reaction-restriction fragment length polymorphism analysis. Folate, vitamin B12, and tHcy concentrations were determined by corpuscle immune chemiluminescence assays. The tHcy concentration was determined using an enzymatic assay. RESULTS: Significant negative correlations (p<0.001) were observed between the serum levels of tHcy and folate (r=-0.252) and vitamin B12 (r=-0.243). Men had significantly higher serum tHcy concentrations than women (p<0.001). Individuals with the MTHFR TT genotype had significantly higher serum tHcy concentrations than individuals with the CC and CT genotypes (p<0.001). The folate level of red blood cells was significantly increased in individuals with the TT genotype than in individuals with the CC genotype (p<0.05). Moreover, in the low vitamin group, the serum tHcy level was significantly correlated with the levels of folate (r=-0.334, p=0.001) and vitamin B12 (r=-0.212, p=0.046). CONCLUSION: The MTHFR C677T polymorphism, folate deficiency, and B12 deficiency were significantly associated with elevated serum tHcy levels. Among these three factors, folate deficiency had the greatest contribution to the serum tHcy concentration, followed by (in order of decreasing effect) MTHFR C677T and vitamin B12 deficiency. Thus, folic acid and vitamin B12 supplementation could help prevent diseases associated with tHcy accumulation, especially in individuals with the MTHFR 677TT genotype.

Conditional gene modification in mouse liver using hydrodynamic delivery of plasmid DNA encoding Cre recombinase.

The success of Cre-mediated conditional gene targeting in liver of mice has until now depended on the generation of Cre recombinase transgenic mice or on viral-mediated transduction. Here, we sought to establish the feasibility of using hydrodynamic gene delivery of Cre recombinase into liver, using a ROSA26 EGFP mouse. The expression of EGFP and beta-galactosidase was exclusively detected in the liver of mice treated with hydrodynamic gene delivery of Cre recombinase, as assessed with fluorescence microscopy and X-Gal staining, respectively; Southern blotting also showed that Cre mediated recombination occurred specifically in the liver and not in other organs. The Cre mediated recombination reached about 61% of hepatocytes of mouse after repeated injection, as analyzed by flow cytometry. These results demonstrate that Cre recombinase can be transferred to the liver of mice through a simple hydrodynamic gene-delivery approach and can mediate efficient recombination in hepatocytes. Thus, hydrodynamic gene delivery of the Cre recombinase provides a valuable approach for Cre-loxP-mediated conditional gene modification in the liver of mice.

Identification, expression pattern, and subcellular location of human RIP isoforms.

XRIP alpha was identified as an adapter protein involved in RAP nuclear import. Several homologs were reported in mammal EST analysis, but the expression pattern and genomic organization of hRIP isoforms were not clarified yet. We isolated nine isoforms of hRIP from a premade human fetal brain library. hRIP alpha is the longest isoform with 219 residues, containing a N-terminal arginine-rich basic region, followed by an acidic region and two C-terminal Zn finger-like structures. hRIP beta deletes one Zn-finger-like structure. Three hRIP alpha isoforms and four hRIP sigma isoforms express truncated proteins due to frame shift. hRIP gamma isoforms lost the C-terminal Zn-finger-like structure. hRIP delta isoforms only contain the N-terminal arginine-rich basic region and the core sequence of the acidic region. The genomic organization of hRIP was identified by bioinformatic analysis. hRIP, containing seven exons, is located at human chromosome 17p13. hRIP was expressed in all 16 detected human tissues with a similar pattern. All EGFP-hRIP fusion proteins were located at the nucleus in the HEK293 cell. The two-polar molecular structure of hRIP might be involved in the basic cell function, and plays a role in the alternative nuclear ingress.

Generation and characterization of transgenic mice expressing tamoxifen-inducible cre-fusion protein specifically in mouse liver.

AIM: To establish transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase specifically in the liver and to provide an efficient animal model for studying gene function in the liver and creating various mouse models mimicking human diseases. METHODS: Alb-Cre-ERt transgenic mice were produced by microinjecting the construct with Cre-ERt fusion gene of DNA fragments into fertilized eggs derived from inbred C57BL/6 strain. Transgenic mice were identified by using PCR and Southern blotting. Expression of Cre-ERt fusion gene was analyzed in the liver, kidney, brain and lung from F1 generation transgenic mice at 8 weeks of age by reverse transcription (RT)-PCR. RESULTS: Four hundred and fourteen fertilized eggs of C57 BL/6 mice were microinjected with recombinant Alb-Cre-ERt DNA fragments, and 312 survival eggs injected were transferred to the oviducts of 12 pseudopregnant recipient mice, 6 of 12 recipient mice became pregnant and gave birth to 44 offsprings. Of the 44 offsprings, two males and one female carried the hybrid Cre-ERt fusion gene. Three mice were determined as founders, and were back crossed to set up F1 generations with other inbred C57BL/6 mice. Transmission of Cre-ERt fusion gene in F1 offspring followed Mendelian rules. The expression of Cre-ERt mRNA was detected only in the liver of F1 offspring from two of three founder mice. CONCLUSION: Transgenic mice expressing tamoxifen-inducible Cre-ERt recombinase under control of the liver-specific promoter are preliminary established.

Transfection of mEpo gene to intestinal epithelium in vivo mediated by oral delivery of chitosan-DNA nanoparticles.

AIM: To prepare the chitosan-pmEpo nanoparticles and to study their ability for transcellular and paracellular transport across intestinal epithelia by oral administration. METHODS: ICR mice were fed with recombinant plasmid AAV-tetO-CMV-mEpo (containing mEpo gene) or pCMVbeta(containing LacZ gene), whether it was wrapped by chitosan or no. Its size and shape were observed by transmission electron microscopy. Agarose gel electrophoresis was used to assess the efficiency of encapsulation and stability against nuclease digestion. Before and after oral treatmant, blood samples were collected by retro-orbital puncture, and hematocrits were used to show the physiological effect of mEpo. RESULTS: Chitosan was able to successfully wrap the plasmid and to protect it from DNase degradation. Transmission electron microscopy showed that freshly prepared particles were approximately 70-150 nm in size and fairly spherical. Three days after fed the chitosan-pCMVbeta complex was fed, the mice were killed and most of the stomach and 30% of the small intestine were stained. Hematocrit was not modified in naive and 'naked' mEpo-fed mice, a rapid increase of hematocrit was observed during the first 4 days of treatment in chitosan-mEpo-fed animals, reaching 60.9+/-1.2% (P<0.01), and sustained for a week. The second feed (6 days after the first feed) was still able to promote a second hematocrit increase in chitosan-mEpo-fed animals, reaching 65.9+/-1.4% (P<0.01), while the second hematocrit increase did not appear in the 'naked' mEpo-second-fed mice. CONCLUSION: Oral chitosan-DNA nanoparticles can efficiently deliver genes to enterocytes, and may be used as a useful tool for gene transfer.

Insulin expression in livers of diabetic mice mediated by hydrodynamics-based administration.

AIM: Transfer and expression of insulin gene in vivo are an alternative strategy to improve glycemic control in type 1 diabetes. Hydrodynamics-based procedure has been proved to be very efficient to transfer naked DNA to mouse livers. The basal hepatic insulin production mediated by this rapid tail vein injection was studied to determine its effect on the resumption of glycemic control in type 1 diabetic mice. METHODS: Engineered insulin cDNA was inserted into plasmid vectors under a CMV promoter, and transferred into STZ induced diabetic mice by hydrodynamic procedure. Glucose levels, body weight of treated mice, insulin levels, immunohistology of the liver, and quantity of insulin mRNA in the liver were assayed to identify the improvement of hyperglycemic complication after plasmid administration. Sleeping Beauty, a transposon system, was also used to prolong the insulin expression in the liver. RESULTS: After plasmid administration, Plasma insulin was significantly increased in the diabetic mice and the livers were insulin-positive by immunostaining. At the same time the hyperglycemic complication was improved. The blood glucose levels of mice were reduced to normal. Glucose tolerance of the treated diabetic mice was improved. Body weight loss was also ameliorated. The rapid tail vein injection did not cause any fatal result. CONCLUSION: Our results suggested that insulin gene could be efficiently transferred into the livers of diabetic mice via rapid tail vein injection and it resulted in high level of insulin expression. The basal hepatic insulin production mediated by hydrodynamics-based administration improved the glycemic control in type 1 diabetes dramatically and ameliorated diabetic syndromes. Hydrodynamics-based administration offers a simple and efficient way in the study of gene therapy for type 1 diabetes.

Conditional gene activation in cultured hepatocytes using a ligand-dependent chimeric Cre recombinase.

By combining liver-specific promoter and a chimeric Cre recombinase, conditional gene activation could be finely achieved in hepatocytes at selected time points. To this end, the expression vector of Cre-ERt under the control of the mouse albumin gene promoter/enhancer, alb-Cre-ERt, was constructed, and transfected into engineering BRL (Rat hepatocytes) and BRK (Rat kidney) reporter cells which carries a chromosomally integrated 'floxed' beta geo gene, which is inserted between the promoter and the human alkaline phosphatase( hAP) reporter gene, thereby preventing hAP reporter gene transcription, respectively. After treatment with 1 micromol/L 4-hydroxytamoxifen(4-OHT), a proportion of hAP staining positive cells were detected by hAP staining. It was further confirmed that 'floxed' beta geo cassette was removed by Cre excision by using PCR analysis of cellular DNA. No background recombinase activity could be detected in the absence of 4-OHT. Moreover, no hAP-positive cells could be detected in BHK cells untreated or treated with 4-OHT. These data suggested that alb-Cre-ERt expression vector was constructed successfully, and 4-OHT could induce Cre-mediated recombination only in hepatocytes expressing Cre-ERt, thereby activating a stably integrated hAP reporter gene. This provides a further foundation for producing transgenic mice expressing such an 4-OHT inducible Cre recombinase specifically in mouse liver.

Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle.

BACKGROUND: Retroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo. METHODS: FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at NheI site of 3' long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2IXm2SN(F) and LMe2IXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and muscle-specificity of factor IX expression with SCID mice. RESULTS: Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24 h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2IXm2SN(R) without changing the orientation of Me2. CONCLUSIONS: LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter.