Expression of human clotting factor Ⅸ mediated by recom- binant lentiviral vector in cultured cells and hemophilia B mice

To explore the expression of human clotting factor Ⅸ (hFⅨ) cDNA in vitro and the feasibility of gene therapy for hemophilia B mice mediated by recombinant lentiviral vector, a recombinant hFⅨ lentiviral vector driven by ubiquitin-C promoter, FUXW, and by ABP liver specific promoter, FAXW, was constructed respectively. Recombinant lentivirus was harvested from 293T cells by calcium phosphate-mediated transient cotransfection of three plasmids (transgene vector, CMV腞8.2, VSV-G). hFⅨ expression was detected in supernatant of 293T, BHK and L-02 cells infected with FUXW virus, whereas higher expression of hFⅨ levels (630 ng/106 cells/48 h) was detected only in L-02 cells infected with FAXW virus. Serum hFⅨ antigen was detected in all hemophilia B mice treated with FAXW virus by tail vein injection, an efficiency level of hFⅨ was observed (45 ng/mL, approximately 1% of normal human levels), the expression lasted for more than 60 d. The results indicated that HIV-based lentiviral vectors offer a promising approach to the gene therapy of hemophilia B.     

Expression of biologically active human clotting factor Ⅸ(hFⅨ) in the mammary gland of transgenic mice

The DNA of human factor Ⅸ (hFⅨ) gene vector pMCⅨm, which had been proven to be able to express in in vitro and living cells, was introduced into 586 zygotes of Kunming White Mice by positive pressure microinjection technique with manual operation. The 499 survival embryos after microinjection were then transferred into pseudopregnant recipient mice and 216 F 0 pups were born. The analysis of PCR and Southern blot hybridization showed that, of the 216, 6 (2 females and 4 males) were integrated with foreign DNA in their genomes, giving an integration frequency of 3% (6/216). Two F\-0 female transgenic mice could express hFⅨ protein in their milk and the content was over 100 ng/mL as measured with ELISA. The biological activities of hFⅨ in the milk of two F\-0 mice were 44 67% and 79 43%, respectively.     

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor Ⅸ in large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25×10¹² particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitro and in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt (2565.76±64.36) ng·(10⁶ cells)^-1·(24 h)^-1 in C2C12 and 453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapy research for hemophilia B, meanwhile, a novel packaging system, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation of industrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated with rAAV-hFⅨ.     

Efficient expression of human factor Ⅸ cDNA in liver mediated by hydrodynamics-based plasmid administration

The transfection of plasmid DNA into mammalian cells is an indispensable tool in the study of gene transfer and gene function. Since the original report in 1990 on the successful expression of a reporter gene in muscle[1], plasmid DNA injection has been widely used to mediate gene transfer study. As a gene transfer vector, naked DNA has many obvious advantages. It is easy and cheap to be prepared and more safe after transfection. But the plasmid mediated gene expression is low because of t…     

Production of transgenic mice carrying green fluorescence proteingene by a lentiviral vector-mediated approach

A pseudo-lentivirus, which carries green fluorescence protein (GFP) expressing cassette, was injected into the perivitelline space of murine fertilized oocytes before transplanting into the oviducts of the foster mothers. The GFP transgenic pups were then obtained. By PCR amplification, fluorescent microscopy and flow assisted cytometry sorting analysis, we found that the integration rate of the transgene was estimated at above 40%. Real-time PCR analysis indicated that the copy number of the integrated GFP cassette was around 40. Fluorescent in situ hybridization analysis demonstrated that the integration pattern was random but inheritable. The transgenic mice with multi-integration sites and various expression levels possessed a great value in practice as well as research. The approach reported herein provides an efficient way to generate and screen the transgenic mouse strains.     

Production of transgenic mice carrying green fluorescence proteingene by a lentiviral vector-mediated approach

A pseudo-lentivirus, which carries green fluorescence protein (GFP) expressing cassette, was injected into the perivitelline space of murine fertilized oocytes before transplanting into the oviducts of the foster mothers. The GFP transgenic pups were then obtained. By PCR amplification, fluorescent microscopy and flow assisted cytometry sorting analysis, we found that the integration rate of the transgene was estimated at above 40%. Real-time PCR analysis indicated that the copy number of the integrated GFP cassette was around 40. Fluorescent in situ hybridization analysis demonstrated that the integration pattern was random but inheritable. The transgenic mice with multi-integration sites and various expression levels possessed a great value in practice as well as research. The approach reported herein provides an efficient way to generate and screen the transgenic mouse strains.     

Expression of PHGPx in mammalian cells and its antiviral effect against coxsackievirus group

To express human phospholipid hydroperoxide glutathione peroxidase (PHGPx) in eukaryotic cells and to study its antiviral effect against Coxsackievirus B3m (CVB3m) in vitro， PHGPx cDNA was amplified from a human testis library using specific primers and cloned into expression vector pcDNA3.1His. Expression of PHGPx was performed in COS-1 cells. The antiviral effect was studied by the treatment of HeLa cells with the recombinant PHGPx. Results showed that the activity of PHGPx expressed in COS-1 cells was 5-fold higher than that in control group, and it inhibited the cytopathic effect on HeLa cells caused by CVB3m. It can be concluded that recombinant PHGPx expressed in COS-1 cells has antiviral effect against CVB3m in vitro.     

Polycistronic strategy for cyanobacterial expression vector construction: Co-transcription of a human gene and a selective marker gene

A polycistronic expression vector, pKGA-NTF1, was constructed for the cyanobacterium. Within this vector, the spectinomycin/streptomycin resistance gene (aadA) facilitated the selection of transformants when co-transcribed with favorite genes. A natural glnA gene was selected as the platform to introduce the plasmid into a neutral site of the Synechococcus sp. PCC 7002 chromosome. Function of the vector was demonstrated by the insertion of a modified human Trefoil factor 3 gene (NTF1) to upstream of the aadA gene and by the analyses of the transformed strains. Antibiotics resistance assays showed that the dicistronic expression cassette conferred high spectinomycin resistance to both the E. coli cells and the Synechococcus cells. PCR analysis and Western-blot analysis were carried out to confirm the integration and expression of the NTF1 gene, respectively. Through simple molecular manipulations, the artificial polycistronic structure described here can be conveniently used to express other favorable genes or operons in cyanobacteria, and to study the cyanobacterial gene expression as well.     

Dual regulatory effects of resveratrol on activation of NF-kB and cell proliferation in human embryonal kidney 293 cells

Resveratrol (3,4 ,5-trihydroxystilbene, Res), a naturally occurring polyphenol, was first detected in grapevines (Vinis vitifera) in 1976[1]. It is also found in various fruits, vegetables and some other plants. It is abundant in grapes and the root of Polygonum cuspidatum, which is an im-portant constituent of traditional Chinese medicine. The concentration of Res in red wine reaches 2×10?6―4×10?5 mol/L. Res has been shown to have antioxidant properties, anti-inflammatory effect, estrog…