Construction of Recombinant Retroviral Vector Containing HIV-1 Tat Gene and Functional Detection of Expressed Tat in Target Cells

Objective: To construct recombinant retroviral vector containing HIV-1 Tatgene and evaluate the junction of the expressed Tat in target cells. Methods: HIV-1 Tat_(101) genewas recovered from pEV plasmid by Hind Ⅲ digestion and cloned into expression plasmid LZESpBMN-Z toconstruct recombinant retroviral expression plasmid named LZRS-Tat_(101). Using the method ofcalcium phosphate, the construct of LZRS-Tat_(101) was then transfected into packaging cell linesPhoenix (ΦNX) which contained env and gal genes encoding structural proteins and pol gene codingfor 3 enzymes ( reverse transcriptase, protease and integrate) essential for retroviral integrationand replication . The stable transfected cell lines was obtained using puromycin to screen for morethan 3 days. Then, immunohistochemical (IHC ) staining was carried out to detect the expressionlevel of Tat_(101) protein in both transiently and stably trancfected ΦNX, respectively. Thesupematants containing recombinant virus collected from transient and stable transfected cells wereemployed to infect 293 cells, respectively, and the expressed Tat in 293 cells was tested by Westernblot. Meantime, the supematants of infected 293 cells was further added to HL3T1 cells which wereHela cell lines containing an HIV-1-LTR/CAT reporter construct to establish a co-culture system.After co-culture for 72 hours, the protein was extracted from HL3T1 cells and used for CAT activityassay. Results: After LZRS- Tat_(101) was transfected into ΦNX, the amount of expressed Tat intransient transfection cells was significantly higher than that in stable transfection cells; Tatcould be detected not only in 293 cells but also in the supematants from 293 cells culture, and Tatin the supematants could activate HIV-1 LTR promoter in HL3T1, resulting in high 'expression of CATlocated at the downstream of LTR. Conclusion: The construct of recombinant retrovirus LZRS-Tat_(101) could express Tat protein in target cells and the expressed Tat was functionally activeand can really exhibit the ability to activate transcription.

Construction of pINC-lacZ Retroviral Vector and its Expression in Mouse Embryonic Stem Cells

A retroviral vector pINC-lacZ containing an E coli β-galactosidase(β-gal)gene was constructed and introduced into the MESPU-13 cells by electroporation.Four G418-resistant colonies were obtained from 1×107,electroporated MESPU-13 cells. Histochemical staining for β-gal activity showed that lacZ gene was expressed in at least three of the four colonies.Southern analysis proved that one copy of foreign gene was integrated in the chromosome of one of the transformed lines(MC15).These results showed that the expression of lacZ gene driven by SiCMVIE promoter can be detected in the transformed MESPU-13 cells.

Transduction of primary rat hepatocytes with bicistronic retroviral vector

INTRODUCTIONHepatocellular transplantation (HCT) could providea therapeutic alternative to orthotopic livertransplantation(OLT) in the treatment of hepaticmetabolic defects and experimental hepaticfailure.Under appropriate conditions,theengrafted liver cells can continue to express liver-specific functions for an indefinite period of time.

Bioactivities of Culture Supernatants from Retroviral Packaging Cells Carrying the Mouse Fas Ligand Gene

The bioactivities of culture supernatants from retroviral packaging cells carrying the mouse Fas ligand (mFasL) gene was investigated. FasLcDNA was cloned into PLXIN with an internal ribosome entry site to link two cistrons through gene recombination technology, PLXIN and the recombinant vector PLFIN were separately transfected into PA317 retrovirus packing cell line by lipofectamine 2000, and the resistant clones were selected with G418 selective medium. The integration of genome DNA was assayed by genomic DNA PCR. NIH3T3 cells were transduced by the culture supernatants from PA317 carrying the mFasLcDNA gene, and were selected with G418 selective medium, so as to select the PLFIN-PA317 clone capable of producing higher titer of supernatants. The levels of mFasL protein on NIH3T3 cells membrane were assayed by flow cytometry (FCM). The biological activity of mFasL on NIH3T3 cells membrane was investigated by the inducing apoptosis of Fas + Yac-1 cells co-cultured with NIH3T3 cells expressing Fas ligand. To explore the direct mFasL cytotoxicity of culture supernatants from retroviral packaging cells carrying the mFasL gene, the culture supernatants from PLFIN-PA317 and PLXIN-PA317 were separately co-cultured with Yac-1 cells in parallel. The recombinant PLFIN was successfully constructed. The highest titer of supernatants from twelve resistant clones was 8.5×10 5 colony-forming-unit (CFU)/ml. The NIH3T3 cells transfected by above supernatants had a higher level of mFasL (53.81±6.9 %), and significantly induced the apoptosis of Fas + Yac-1 cells (56.78±4.5 %), as both were cocultured for 5 h at 1∶1 ratio, whereas it is 7.08±3.4 % in control group (P<0.01). Supernatant from PLFIN-PA317 could also directly induce the apoptosis of Yac-1 within 5 h of incubation. Thus, the culture supernatants from PLFIN-PA317 possessed both infectivity and cytotoxicity of mFasL.

PRELIMINARY STUDY OF RETROVIRAL MEDIATED TRANSFER OF THE HUMAN mdr-1 GENE INTO MURINE AND HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS

To investigate the characteristics of multidrugresistance and transplantation of modified stem/ progenitor cells by multidrugresistant gene (mdr1 gene), we established PA317/MDR1 cell line which producing retroviruses by transfecting the retroviral vector PHaMDR1/A into packging cell line PA317 by Lipofectin. The virus titer of the supernatants was 1.2×105 cfu/ml. We transfected the murine hematopietic cells collected from 5FU pretreated mice and they showed the ability to reconstitute the longterm hematopoiesis of preirradiated mice. After 4 months, both of bone marrow cells and peripheral blood cells of transplanted mice still contained mdr1 gene. We also transfered mdr1 gene into human bone marrow CD34+ cells selected by using magnetic cell sorting system. PCR analysis showed that transduced CD34+ cells maintained the mdr1 cDNA. A fraction of CFUGM originated from transfected CD34+ cells had the charactor of resistance to Taxol. It is indicated that mdr1 gene can be transduced into murine and human stem/proginitor cells through retroviral mediated gene transfer and it protects the transfected cells from cytotoxic drugs.

Construction and expression of retroviral vector pLEGFP-N1-TERT in preparation of seed cells for skin tissue engineering

Objective:To construct the retroviral vector pLEGFP-N1-telomerase reverse transcriptase(TERT)and to investigate the expression of TERT in neonatal mouse bypodermal cells.Methods:The polymerase chain reaction(PCR)-amplified TERT gene was inserted into plasmid pLEGFPN1.The positive clone was identified by restriction enzyme digestion and sequencing,then was transfected into packaging cells to produce retrovirus particles.Neonatal mouse hypodermal cells were infected with the virus to generate a stable cell line.The TERT mRNA expression level,telomerase activity,and enhanced green fluorescent protein(EGFP)expression level were analyzed.Results:Retroviral vector pLEGFP-N1-TERT was constructed successfully,and a stable cell line of neonatal mouse hypodermal cells expressing EGFP was established.Western blot and immunohistochemical assay showed that the expression level of TERT was significantly elevated in the neonatal mouse hypodermal cells.Conclusions:A high titer of retrovirus pLEGFP-N1-TERT mediates high-level expression of the exogenous TERT gene in the neonatal mouse hypodermal cells.This protocol has potential applications for skin tissue engineering and cell transplantation therapy.

CONSTRUCTION AND IDENTIFICATION OF RETROVIRAL VECTOR EXPRESSING HUMAN INTERLEUKIN-17 GENE

Human interleukin 17(IL17) is a newly found cytokine secreted by activated T lymphocytes. Many of its characteristics remain unknown. To provide a way for understanding its role in immunology and hematology further, we constructed a recombinant retroviral vector pL17SN containing the human IL17 gene about 1.3kb with the coding region. The constructed retroviral vector packaged in CRIP cells could infect human primary fibroblasts, and could stimulate the primary fibroblasts secreting human GMCSF and IL6. The retroviral vector containing the human IL17 gene we constructed may present an efficient way to understand the biological functions of human IL17 and to investigate applications of IL17 in cancer gene therapy.

Expression of the Capsid Precursor Protein gene of Foot-and-mouth Disease Virus and Green Fluorescent Protein Gene in BHK-21 Cells Mediated by Retroviral Vector

We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.

THE ENHANCED GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN TUMOR CELLS LABELLED BY RETROVIRAL TRANSDUCTION

Objective: To investigate the feasibility of marking the human tumor cells with enhanced green fluorescent protein (EGFP) in vitro. Methods: The retroviral vector LGSN encoding EGFP was constructed and three human tumor cell lines were infected with LGSN amphotropic virus. Tumor cell lines that stably express EGFP were selected with G418. The integration and expression of EGFP gene were analyzed by polymerase chain reaction, and flow cytometry (FCM). Results: After gene transfection and ping-pong transduction, amphotropic producer line Am12/LGSN was generated with a stable green fluorescence signal readily detectable by FCM in up to 97% of examined cells. The viral titer in the supernatants was up to 8.2×105CFU/ml. After transduction and selection, G418-resistant leukemia K562, mammary carcinoma MCF-7, and bladder cancer 5637 cells were developed, in which the integration of both EGFP and neomycin resistance gene was confirmed by DNA amplification. In comparison with uninfected cells, FCM analysis revealed EGFP expression in up to 90% (range 85.5%–90.0%) of tumor cells containing LGSN provirus. Conclusion: The retroviral vector LGSN can effectively mark the human tumor cells with a stably EGFP expression which may be in studying tumor growth, metastasis and angiogenesis.

Construction of retroviral vectors to induce a strong expression of human interferon-β gene in human hepatoma cells

Establishing the hepatoma cell-specific expression of human interferon gene mediated by retroviral vectors. Methods: Human interferon-β complementary DNA (IFN-β cDNA) was inserted into polylinker site of pMNSM retroviral vector to construct recombinant retroviral vector pMNSIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter, and MNAIFNB, where the transcription of IFN-β gene was driven by SV40 early region promoter regulated by α-fetoprotein enhancer. The retroviral constructs were respectively introduced into PA317 amphotropic packaging cells by means of lipofectamine mediated gene transfer procedure. The plasmids transfection efficiency was among (4-25)ｘ103 colonies/μg DNA/106 PA317 cells. The retrovirus infection efficiency was among (4. 5-500)ｘ104 Colony Forming Units (CFU)/ml. The recombinant retroviruses were used to infect human hepatoma cells, renal cell carcinoma cells and melanoma cell lines in the presence of 4 μg/ml polybrene. Results: Dot hybridization of total RNA from the neomycin resistant colonies and interferon expression assay indicated that human α-fetoprotein enhancer induced efficient and specific transcription and expression of IFN-β gene driven by the promoter of different origin in human hepatoma cells by which α-fetoprotein was highly produced. Conclusion: Cis-active element of α-fetoprotein gene can drive IFN-β gene specifically expressed in human hepatoma cells, which presents some valuable materials for the hepatoma-specific immune gene therapy.

Construction and identification of a retroviral vector of bcl-2 mRNA-cleaving ribozyme gene

The bcl-2 mRNA-cleaving ribozyme (RZ) gene was synthesized with EcoRI and BamHI sites, each on one of the terminals. The synthesized gene was cloned to the HincII site of the plasmid pGEM-3Zf(-) and the recombinant plasmid was termed 3ZRZ (+)/(-). The gene was sequenced and found to be correct, and was used for in vitro transcription and in vitro cleaving of bcl-2 mRNA. The RZ gene was cut out from the plasmid and subcloned in a retroviral vector pDOR-neo at the BamHI and EcoRI sites, and the new recombinant retroviral vector was named pDOR-RZ.

Improvement in Socio-Economic Productivity of HIV Positive Individuals on Antiretroviral Treatment in a Private Setting in South India

PLHIV have decreased economic productivity both due to direct and indirect causes. Data from developed countries have shown that at the societal level, high costs ART are offset by increased productivity. We hypothesized that post-ART the SES would improve regardless of the baseline SES and will be sustained over time. Our objective was to perform a comprehensive SES evaluation pre/post ART initiation using an ambispective cohort study design. We used Indian household-specific SES validated tool, with score of 76 being affluent, along with clinical, ART adherence data at median of 6 and 18 months post ART, and compared using paired t-tests. Among 140 persons started on ART, with a median follow up of 22 months, 118 had Pre-ART SES data, of these: 57% were women;median age was 38 years;67% were married;89 (78%) had heterosexual sex as HIV risk;40 (34%) had major OI and/or TB at presentation. Reported self-occupation was: skilled labourers 41 (35%);12 (10%) unskilled labourers;27 (23%) housewives;26 (22%) pro-fessionals/blue collar job;1 student, 10 unemployed. The median pre-post ART CD4 cell counts were: 187 and 454 cells/cumm (P < 0.01);median body weight pre-post ART was 54 and 57 kg (P < 0.01);97% of the participants were 100% adherent. The mean Pre-ART total SES score was 37.06 (+/-10.2);and Post-ART SES score 40.62 (+10.1 P < 0.001) and these results were sustained over time and remained significant even when only monthly income was considered. Our data show a significant impact of ART on SES in a sustained manner in a developing world setting, which has policy level implications.

High level expression of human Factor Ⅷ in mammalian cells after retroviral-mediated gene transfer

Abstract:Objective To develop a retroviral-mediated high efficient expression system of human coagulation factor Ⅷ. Methods The LNC-FⅧBD retroviral vector was generated by cloning a human B-domain-deleted (760aa～1639aa) Factor Ⅷ (FⅧ) cDNA (FⅧ cDNA BD) into the retroviral vector pLNCX. Several mammalian cell lines, including NIH3T3, CHO, Cos-7 and human hepatic cell line, L-02, were transduced with viral supernatant from the highest virus-producing PA317 clone. Antigen and coagulant activity of human FⅧ in cell culture medium were measured by ELISA and a one-stage method, respectively. RT-PCR was performed for the detection of FⅧBD mRNA. Results Human FⅧ was expressed in all four target cells, with the highest FⅧ expression observed in NIH3T3. The coagulant activity of secreted FⅧ was up to 1.6U/106 cells*24?hrs-1, and the FⅧ antigen was 500?ng/106 cells*24?hrs-1. FⅧ coagulant activity and antigen expressed by transduced CHO cells were 0.12?U/106 cells*24?hrs-1 and 62.4?ng/106 cells*24?hrs-1, respectively. Human FⅧ expression was relatively low in Cos-7 and L-02 cells. RT-PCR results demonstrated transcription of FⅧcDNA BD in the target cells.Conclusions The constructed retroviral vector was able to direct high level expression of human FⅧ in various mammalian cell lines. It has potential utility in the future gene therapy for Hemophilia A.

Retroviral vector containing human p16 gene and its inhibitory effect on Bcap-37 breast cancer cells

Objective To study the effects of the p16 gene on tumor cell growth inhibition and cell cycle arrest. Methods The recombinant retroviral vector pLp16SN was constructed by cloning the human p16 cDNA into the retroviral vector pLXSN. Retroviruses with or without the p16 gene were obtained by transfecting pLp16SN and pLXSN vectors into PA317 cells. Bcap-37 human breast cancer cells were infected with these retroviruses followed by selection with G418. The expression of p16 was detected by Northern and Westem blots. Cell biological characteristics, including cell growth rate, cell cycle and tumorigenesis in nude mice were assessed.Results Both mRNA and protein expression of p16 in Bcap-37 cells transfected with retroviral vector containing the p16 gene were much higher than that in Bcap-37 cells transfected with empty vector or parental Bcap-37 cells. Cell overexpressing the p16 gene exhibited a slower rate of growth, a higher percentage of cells in the G1 phase, and smaller tumors in nude mice, compared with parental Bcap-37 cells and cells transfected with empty vector.Conclusion Overexpression of the p16 gene could suppress the growth of Bcap-37 breast cancer cells by arresting the cell cvcle at the G1 to S-phase.

Effects of recombinant retroviral vector mediated human insulin like growth factor-1 gene transfection on skeletal muscle growth in rat

Background This study transferred a recombinant gene encoding human insulin like growth factor-1 （hIGF-1） into modified primary skeletal myoblasts with a retroviral vector （pLgXSN） and determined whether the hIGF-1 promoted growth of skeletal muscle in rat.Methods hlGF-lcDNA was amplified in vitro from normal human liver cells by using RT-PCR and cloned into plasmid vector pLgXSN. The recombinant vector pLghIGF-1SN and control vector pLgGFPSN were transfected into packaging cell PT67 and G418 was used to select positive colony. Myoblasts were infected with a high titre viral supernatant and transduction efficiency was evaluated as GFP expression. The expression of hIGF-1 mRNA in myoblasts was investigated by immunocytochernistry and RT-PCR. MTT assays detected the growth of myoblasts in vitro. Myoblasts transduced with pLghlGF-1SN were injected into hind limb muscles of 10-12 week male SD rats. Formed tissues were harvested 4 weeks later. Myocyte diameter, mean weight of hind limb and body were measured to evaluate the skeletal muscle growth. Results Recombinant retroviral plasmid vector pLghlGF-1SN was constructed successfully. The titre of the packaged recombinant retrovirus was 1 × 106 cfu/ml. The transfection rate of PT67 cells reached 100% after G418 screening, hIGF-1 expression was positive in myoblast-IGF-1. The proliferation rate of myoblast-IGF-1 in vitro was higher than GFP-myoblast or myoblast （P〈 0.05）. The mean weights of hind limb and body of rats injected myoblast-IGF-1 were higher than those of the rats injected with myoblast-GFP or myoblast （P〈 0.05）. Myocyte diameter had a significant increase in IGF-1 group compared to GFP group and myobiast group （P〈 0.05）. Conclusions The transfection of the human IGF- 1 gene mediated by a retroviral vector can promote the growth of skeletal muscle in rats. Genetically modified primary skeletal myoblasts provide a possibly effective approach to treat some skeletal muscle diseases.

Anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection

Background This study was designed to obtain a recombinant retroviral vector containing the human hepatocellular carcinoma-related gene ANGPTL4 (angiopoietin-like 4) cDNA and to evaluate the anti-tumor effect of recombinant retroviral vector-mediated human ANGPTL4 gene transfection.Methods ANGPTL4 cDNA was cloned in vitro from normal human liver cells HL-7702 by using RT-PCR, and then subcloned into the plasmid vector pMSCV and sequenced. The retroviral plasmid vectors pMSCV-ANGPTL4, pVSV, and pGAG-POL were co-transfected into the packaging cell line 293 EBNA under mediation of lipofectamine. A high-titer retrovirus was obtained as a result, and HepG 2 cells were infected with this retrovirus in vitro. Flow cytometry and fluorescence microscopy were used to detect expression of green fluorescence protein (GFP). The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was investigated using RT-PCR. The formation of tumors in nude mice and MTT assays were used to detect the growth of HepG 2-ANGPTL4 cells in vivo and in vitro, respectively.Results The cDNA sequence of the cloned ANGPTL4 gene was consistent with the recently reported sequence. Thus, the recombinant retroviral vector pMSCV-ANGPTL4 was constructed successfully. The titer of the packaged recombinant retrovirus was 1.4×106 infective viral grains/ml, and the rate of HepG 2-ANGPTL4 cells expressing GFP was 68.45%, with an average intensity of fluorescence 31.67 times greater in HepG 2-ANGPTL4 cells than in HepG 2 cells. The expression of ANGPTL4 mRNA in HepG 2-ANGPTL4 cells was higher than in HepG 2-pMSCV cells (154% higher) or HepG 2 cells (161% higher). The proliferation rate of HepG 2-ANGPTL4 cells in vitro was obviously lower than those of HepG 2-pMSCV cells and HepG 2 cells (P<0.01). The mean volume and weight of tumors seeded from HepG 2-ANGPTL4 cells were obviously lower than the mean volume or weight of tumors seeded from HepG 2 cells and HepG 2-pMSCV cells (P<0.01).Conclusion A stable ANGPTL4-transfected human liver cancer cell line HepG 2-ANGPTL4 has been created. The transfer of the human ANGPTL4 gene mediated by a retroviral vector is a possibly effective approach for liver cancer therapy.

HIGH EFFICIENT TRANSFER AND EXPRESSION OF HUMAN CLOTTING FACTOR Ⅸ cDNA IN CULTURED HUMAN PRIMARY SKIN FIBROBLASTS FROM HEMOPHILIA B PATIENT BY RETROVIRAL VECTORS

To study the possibility of somatic gene therapy for hemophilia B via gene transfer to primary factor Ⅸ-deficient skin fibroblasts, we constructed four retroviral vectors containing factor Ⅸ cDNA driven by retroviral LTR promoter, SV40 early promoter and mouse MT-I promoter, respectively. These retroviral vectors were transfected into an amphotropic packaging cell line, PA317 cells, by electroporation, and a human iibrosarcoma cell line, HT1080 cells, was used to assay the factor Ⅸ-virus titers of these four virus-producing PA317 cells, which ranged from 2×10~4 to 5×10~5 cfu/ml. The factor Ⅸ proteins produced by bulk population of four virus-producing PA317 cells were determined by ELISA. Results showed that LTR promoter directed the highest production of factor Ⅸ at the rate of 584 rig/10~6 cells/24h, while SV40 early promoter and MT promoter directed about 10 and 20 times less production of factor Ⅸ than LTR promoter. The highest expressed retroviral vector XL-Ⅸ was used to infect a line of factor Ⅸ-deficie human primary skin fibroblasts, FDⅨ cells. The factor Ⅸ secretion rate of the infected FDⅨ cells was about 549 ng/10~6 cells/24h and over 75% of secreted factor Ⅸ was biologically active. We are convinced that this factor Ⅸ-deficient human primary skin fibroblast had been cured, or genetically corrected, by retroviral-mediated gene therapy in vitro.

Mesenchymal stem cells transduced by PLEGFP-N1 retroviral vector maintain their biological features and differentiation

Background Enhanced green fluorescent protein （EGFP） has been an important reporter gene for gene therapy. Human mesenchymal stem cells （hMSCs） are ideal target cells in cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated by PLEGFP-N1 retroviral transduction.Methods hMSCs were isolated from human bone marrow by density gradient fractionation and adherence to plastic flasks. Individual colonies were selected and cultured in tissue dishes. Packaging cells PT67 were transfected by PLEGFP-N1 retroviral vector , and hMSCs were transduced by viral supernatant infection. Meanwhile, hMSCs-EGFP were identified by immune phenotypes and whether it could differentiate into osteoblasts or adipocytes under conditioned media was investigated.Results The rate of stably transduced hMSCs-EGFP was up to 96% after being screened by G418. hMSCs-EGFP exhibited fibroblast-like morphological features. Flow cytometric analyses showed that hMSCs-EGFP were positive for CD73, CD105, CD166, CD90 and CD44, but negative for CD34 and CD45. In addition, it could functionally be induced into osteocytes or adipocytes under conditioned media. These biological features of hMSCs-EGFP were consistent with those of hMSCs.Conclusions hMSCs transduced by PLEGFP-N1 retroviral vector can be used in vivo securely because they can maintain their biological characteristics and differentiation. It is a simple and reliable way to trace the changes of hMSCs in vivo by EGFP during cell transplantation and gene therapy.

Human clotting factor Ⅸ (hF Ⅸ ) secretion in goat milk after direct transfer with hF Ⅸ minigene into mammary gland by using retroviral vectors

THE mammary gland bioreactor system of lactating animal, established by germ-linemicroinjection and embryo transplantation, has been one of the most exciting projects in thefield of biotechnology. However, this system is costly, laborious and tim-cosuming. In 1994,Johanna et al. reported that secretion of a foreign protein, human growth hormone (hGH),in milk had been achieved after direct introduction of the cDNA into the mammary gland ofgoats by replication-defective retroviral vector. In that work, the in vivo expression of

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 LMe2IXm_2SN(F) and LMe2IXm_2SN(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/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2IXm_2SN(R) produced much less FIX in vivo in SCID mice than LMe2IXm_2SN(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.