Expression of human factor IX gene in murine plasma through lentiviral vector-infected haematopoietic stem cells

1. Haematopoietic stem cells (HSC) are an attractive target for gene therapy. Gene transfer to HSC can provide a potential cure for many inherited diseases. Moreover, recombinant lentiviral vectors can transfer genes efficiently to HSC. In the present study, we used the recombinant lentiviruses FUGW (Flip, ubiquitin promoter, GFP and WRE vector) and FUXW (Flip, ubiquitin promoter, F IX and WRE vector), which carry the enhanced green fluorescent protein (EGFP) and human factor IX (hFIX) gene, respectively, to infect HSC. 2. High titres of recombinant lentivirus were prepared from 293T cells by calcium phosphate-mediated transient cotransfection. Murine mononuclear cells (MNC) separated from murine bone marrow and HSC separated by magnetic cell sorting were cultured in vitro. Cells they were infected by the recombinant lentiviruses FUGW and FUXW. The expression of EGFP was observed under a fluorescent microscope and was analysed by fluorescence-activated cell sorting, whereas the expression of hFIX was detected by ELISA. 3. The results show that the lentiviral vectors can efficiently infect murine HSC in vitro and that transduction was more efficient following cytokine treatment with interleukin (IL)-3, IL-6 and stem cell factor. 4. Haematopoietic stem cells infected with lentivirus FUXW were transplanted into [(60)Co]-irradiated non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. The expression of hFIX in the blood plasma of the transplanted mice reached a peak of 44.9 +/- 7.6 ng/mL on Day 7. An assay of transaminase levels and a histological study of the liver showed that there was no significant damage following HSC transplantation to mice. 5. The results of the present study suggest that transplantation of HSC results in the persistant expression of hFIX in mice, which may be useful in haemophilia B gene therapy.     

Conditions affecting hydrodynamics-based gene delivery into mouse liver in vivo

1. It has been demonstrated that the hydrodynamics-based procedure has high efficiency to deliver foreign genes into the liver. The widespread use of this procedure in gene function studies and as a treatment option for liver and other organ diseases puts considerable importance on the investigation of various conditions that affect hydrodynamics-based gene delivery into mouse liver in vivo. 2. Various conditions, including the volume, speed and solution of the injection and the state, gender and strain of the animal were manipulated to evaluate their effect on the expression levels in mice of human factor IX (hFIX) 8 h after tail vein injection of the plasmid pCMV-hFIX. 3. It was found that an injection volume of 2-2.5 mL and an injection speed of 5-7 s were very effective in delivering DNA into the mouse liver. Using Ringer's solution as an injection fluid increased the efficiency of hFIX expression. 4. Anaesthetized mice expressed higher hFIX than conscious mice. Males expressed higher hFIX than females. The ICR mouse strain demonstrated higher expression of the foreign gene than did the C57 strain. 5. The effects of these specific factors on hFIX expression may be caused by variations in hydrostatic pressure, the degree of liver damage and liver size. 6. It can be concluded that there are optimal conditions for hFIX expression in the liver. This information may be helpful for the application of hydrodynamics-based procedures in the investigation of gene expression and gene therapy.     

重组腺相关病毒2型/人凝血因子IX的质量研究重组腺相关病毒2型/人凝血因子IX的质量研究

目的研究并建立重组腺相关病毒2型/人凝血因子IX(recombinant adeno-associated virus type 2/human blood coagulation factor IX,rAAV-2/hFIX)的质量标准。方法采用PCR法确认病毒所携带的重组核酸结构和测定辅助病毒(helper virus)和野生型腺相关病毒（wtAAV）的残留片段。SDS-PAGE电泳测定病毒外壳蛋白分子量及纯度，TSK gel SP-NPR阳离子交换柱系统测定病毒颗粒纯度。以斑点杂交法测定病毒颗粒数。一期法于IX因子基因剔除小鼠体内测定rAAV-2/hFIX生物学活性，并通过ELISA法测定感染BHK-21细胞后hFIX的表达量。结果PCR法确证病毒的重组核酸结构与构建预期相同；在1×10⁷ VG的rAAV-2/hFIX颗粒中，残留辅助病毒的基因片段数少于1个拷贝；在1×10⁸ VG的rAAV-2/hFIX颗粒中，野生型AAV-2基因片段数少于1个拷贝。病毒颗粒及外壳蛋白纯度均大于98%，病毒颗粒数大于1.0×10₁₅VG·L^-1(virus genome·L^-1)。IX因子剔除小鼠肌肉注射病毒后21 d，小鼠血液中人凝血因子IX活性达到大于正常人因子IX活性的15%，IX因子的体外表达水平大于20.0 μg·L^-1。其他各项检测指标均符合规定。结论建立了rAAV-2/hFIX的质量标准，用于控制产品质量。     

Enhancement of naked FIX minigene expression by chloroquine in mice

INTRODUCTIONDevelopment of nonviral naked DNA transfectionis attractive since they have several advantages overviral-based vectors, eg, simplicity of construction, easeof large-scale production, cost effectiveness, less toxic,nonimmunogenic and the introduced exogenous genesdo not integrate into the host genome. However, therelatively low and transient nature of gene expressionlimited the application of naked DNA transfer system.Liu[1] et al and Zhang[2] et al have developed a tech-niq…     

A New Potent hFIX Plasmid for Hemophilia B Gene Therapy

PURPOSE: The purpose of this work was to construct and characterize a new potent hFIX plasmid, p2SV-hFIX, which has two hFIX expression units containing the SV40 promoter/enhancer for hemophilia B gene therapy. METHODS: p1SV-hFIX was constructed by insertion of amplified hFIX cDNA at the ECORI and Xbal sites of pSI expression vector containing simian virus 40 (SV40) promoter/enhancer. To construct p2SV-hFIX, the hFIX expression cassette was isolated from p1SV-hFIX by digestion with restriction enzymes, and the purified expression cassette was inserted at the BglII site of another plSV-hFIX. The gene expression of p1SV-hFIX, p2SV-hFIX, and a plasmid containing a liver-specific apoE enhancer and alpha antitrypsin promoter, pAAV-hAAT-hFIX. were evaluated in various cell lines using polyethylenimine (PEI) as a gene carrier in vitro. RESULTS: The construction of p1SV-hFIX and p2SV-hFIX were confirmed by restriction enzyme studies. The transfection efficiency of p2SV-hFIX was 3.83-fold and 7.16-fold higher than that of pAAV-hAAT-hFIX in C2C12 and NIH3T3 cells, respectively. p2SV-hFIX also showed higher transfection efficiency than p1SV-hFIX in both cells. CONCLUSIONS: In accordance with these results, p2SV-hFIX is a new potent hFIX plasmid that can be transfected in various cells. Systemic delivery of p2SV-hFIX via intravenous or intramuscular injection is feasible for treatment of hemophilia B.     

In utero exposure to di-(2-ethylhexyl) phthalate affects liver morphology and metabolism in post-natal CD-1 mice

The plasticizer di-(2-ethylhexyl)phthalate (DEHP) affects reproductive development, glycogen and lipid metabolism. Whereas liver is a main DEHP target in adult rodents, the potential impact on metabolic programming is unknown. Effects of in utero DEHP exposure on liver development were investigated upon treatment of pregnant CD-1 mice on gestational days (GD)11–19. F1 mice were examined at post-natal days 21 (weaning) and 35 (start of puberty): parameters included liver histopathological, immunocytochemical and α-fetoprotein (AFP) gene expression analyses. In utero DEHP exposure altered post-natal liver development in weanling mice causing significant, dose-related (i) increased hepatosteatosis, (ii) decreased glycogen storage, (iii) increased β-catenin intracytoplasmic localization (females only). At puberty, significantly decreased glycogen storage was still present in males. A treatment-induced phenotype was identified with lack of glycogen accumulation and intracytoplasmic localization of β-catenin which was associated with increased AFP gene expression. Our findings suggested that DEHP alters post-natal liver development delaying the programming of glycogen metabolism.     

Liver site-specific gene transfer following the administration of naked plasmid DNA to the liver surface in mice

The present study was undertaken to investigate liver site-specific gene transfer following the administration of naked plasmid DNA (pDNA) to the liver surface in mice. We examined whether genes could be delivered to the liver site specifically by utilizing the glass-made diffusion cell that is able to limit the contact dimension between the liver surface and pDNA solution administered. Gene expression was detected at the site of diffusion cell attachment (site 1) and was significantly higher than in other liver sites and tissues. Moreover, gene expression was also detected at deeper site from the liver surface (noncontact side with pDNA solution). The level of gene expression at site 1 did not change significantly with pDNA treatment for 10, 30, and 60 min. In conclusion, we demonstrated that naked pDNA administered to the liver surface in mice was taken up from its surface, and subsequently the protein encoded by pDNA could be produced site specifically.     

四氯化碳损伤小鼠肝脏基因表达谱的变化四氯化碳损伤小鼠肝脏基因表达谱的变化

目的研究CCl₄肝损伤小鼠肝脏的基因表达谱，筛选与CCl₄肝损伤相关的基因网络，探讨其肝损伤机制。方法提取小鼠的肝组织总RNA，经反转录分别用Cy3和Cy5荧光标记，制备用于芯片杂交的cDNA探针；cDNA探针与联合基因公司BioStarM-141S小鼠基因表达谱芯片进行杂交，结果由扫描仪扫描并用软件进行分析统计。结果 在CCl₄肝损伤小鼠的基因表达谱中，有379条基因发生了差异性表达(2.69％)。其中，163条基因表达量明显上调，另外216条基因表达量明显下调。结论CCl₄引起小鼠肝脏基因表达谱变化，利用小鼠基因表达谱芯片能大规模、高通量筛选与CCl₄肝损伤相关基因，对进一步阐明CCl₄及类似的化学肝毒物对肝脏的损伤机制有十分重要的意义。     

Direct transfer of A20 gene into pancreas protected mice from streptozotocin-induced diabetes

AIM: To investigate the efficiency of transfer of A20 gene into pancreas against STZ-induced diabetes. METHODS:PVP-plasmid mixture was directly transferred into the pancreatic parenchyma 2 d before STZ injection. The uptake of plasmid pcDNA3-LacZ or pcDNA3-A20 was detected by PCR and the expression of LacZ was confirmed by histological analysis with X-gal. A20 expression in the pancreas of pcDNA3-A20 transgenic mice was measured by RT-PCR and Western blots. Urine amylase, NO generation, and histological examination were examined. RESULTS:Injection of PVP-plasmid mixture directly into the pancreatic parenchyma increased urine amylase concentration 16 h after operation and reversed it to nearly normal 36 h later. On d 33 LacZ expression could be found in spleen,duodenum, and islets. The development of diabetes was prevented by direct A20 gene transferring into the pancreas and A20-mediated protection was correlated with suppression of NO production. The insulitis was ameliorated in A20-treated mice. CONCLUSION: Injection of PVP-plasmid mixture directly into the pancreatic parenchyma led to target gene expression in islets. Direct transfer of A20 gene into the pancreas protected mice from STZ-induced diabetes.     

施普睿达对荷H22肝癌小鼠基因表达谱的影响

目的研究苦豆子生物碱衍生物施普睿达对荷H22肝癌小鼠基因表达谱的影响,探讨施普睿达抗肝癌的分子机制。方法应用包含4096个小鼠基因的cDNA芯片检测施普睿达对荷H22肝癌小鼠基因表达谱的影响,并对差异基因进行GO聚类和统计分析。结果施普睿达治疗前后共有358个基因出现差异表达,83个基因的表达差异达到3倍以上,表达差异在2倍以上的基因共有275个。经GO分析,差异表达基因包括许多与细胞凋亡、细胞周期、代谢、免疫应答和DNA修复等相关的基因。结论施普睿达主要是通过调控肿瘤细胞周期进程、影响代谢和免疫应答、调节肿瘤细胞凋亡相关基因的表达等多种途径抑制肿瘤的增殖和转移。     

Pharmacokinetics and in vivo gene transfer of plasmid DNA complexed with mannosylated poly(L-lysine) in mice

To achieve mannose receptor-mediated, cell-specific, in vivo gene transfer by intravenous injection of plasmid DNA, mannosylated poly(L-lysine) (Man-PLL) was synthesized as a carrier molecule, and mixed with a plasmid DNA encoding chloramphenicol acetyltransferase (CAT) gene to form DNA/Man-PLL complex. The particle size and zeta potential of DNA/Man-PLL (prepared at 1:0.7 on a weight basis) were determined to be 220 nm and +12 mV, respectively. The pharmacokinetics of the DNA/Man-PLL complex was assessed in mice using 32P-labeled DNA ([32P]DNA). After intravenous injection of [32P]DNA/Man-PLL, the radioactivity in plasma fell rapidly and was recovered mainly in the liver nonparenchymal cells. The amount in the liver reached more than 80% of the dose. Radioactivity observed in kidney, lung, and spleen was very low compared to that in the liver. Then, the in vivo gene expression after intravenous injection of DNA/Man-PLL was examined by a CAT assay. Highest CAT activity was detected in the liver, but no activity was detected in the lung, kidney, and spleen. These results clearly indicate that a cell-specific gene delivery system can be developed by regulating the biodistribution of DNA/carrier complex through the control of its physicochemical properties.     

Analysis of metabolic and gene expression changes after hydrodynamic DNA injection into mouse liver

The hydrodynamic injection in mice tail vein of a plasmid (40 μg DNA) bearing the human α1-antitrypsin gene mediates: a) good liver gene transfer resulting in therapeutic plasma levels of human protein (1 mg/ml, approximately) from days 1-10 after injection; b) low liver injury as demonstrated by a poor and transient increase of aspartate aminotransferase (AST) and alanine transaminase (ALT) in mouse plasma; 3) limited expression and metabolic changes in host liver genes and metabolites as evaluated on days 2 and 10 after injection. Groups of three mice were uninjected (control) or hydrodynamically injected with saline or plasmid DNA and then sacrificed on days 2 and 10 after injection. The results of principal component analysis (PCA) show, both in expression microarray and metabolomic analysis, that changes between control and hydrodynamically injected groups are not dramatic and tend to normalize after 10 d. The differences are even smaller between DNA and saline hydrodynamically injected mice. Hydrodynamic injection induces a complex but limited gene expression and metabolic change which includes variations in molecules related to energy metabolism and stress response. The results contribute to support that hydrodynamic method is a safe procedure of liver gene transfer but the long-term effect of hydrodynamic gene transfer procedure, remains to be studied.     

pcDNA3.1(+)/GDF-5真核表达质粒的构建及其在小鼠骨髓基质干细胞的表达

目的:通过基因重组技术体外构建真核表达质粒pcDNA3．1( )／GDF-5,并检测其在小鼠骨髓基质干细胞中的表达。方法:提取孕14 d小鼠胚胎肢芽组织总RNA,RT-PCR扩增,将扩增产物GDF-5基因片断插入至pcDNA 3．1( )载体,并进行酶切鉴定及测序;脂质体介导pcDNA 3．1( )／GDF-5重组质粒瞬时转染小鼠MSC,RT-PCR和免疫细胞化学检测GDF-5的表达。结果:重组质粒双酶切图谱显示有5．4 kbp和1．6 kbp两条带;测序结果与Genbank中的序列完全相同;转染后RT- PCR显示实验组有一219bp特异性条带,免疫细胞化学检测发现实验组细胞胞浆内有棕色阳性染色,实验对照组和空白组均为阴性。结论:本实验成功构建pcDNA3．1( )／GDF-5真核表达质粒,转染小鼠MSC中有GDF-5表达,为进一步研究其在软骨发育机制和软骨骨组织工程领域提供了实验基础。     

Evaluation of long-term gene expression in mouse liver using PhiC31 integrase and hydrodynamic injection

PhiC31 integrase has the potential to achieve long-term transgene expression because of site-specific and unidirectional recombination. In this study, plasmid DNA (pDNA) encoding Gaussia luciferase (Gluc) cDNA was constructed and the optimal condition for long-term gene expression using phiC31 integrase in mouse liver after hydrodynamic injection was investigated. Gluc is secreted and thus allows quantification of its expression level in blood and easy determination of the expression time-course. Mice were co-transfected with 25?μg donor pDNA (pORF-Gluc/attB) and different amounts of helper pDNA (pCMV-int; from 1 to 50?μg). Serum Gluc expression was evaluated during 120?d. The most highly sustained gene expression was obtained when 10?μg of helper pDNA was co-transfected in ICR and C57BL/6 mice. However, the Gluc expression in C57BL/6 mice was slightly lower and less durable than that in ICR mice. Little hepatic damage caused by phiC31 integrase was observed during 120?d in ICR and C57BL/6 mice.     

Particle-mediated gene transfer of murine interleukin-12 cDNA suppresses the growth of Lewis lung carcinoma

We evaluated the effectiveness of the Helios gene gun system, a recently developed, commercially available gene gun device. Following skin transfection with beta-galactosidase or interleukin-12 cDNA using the gene gun, beta-galactosidase expression was detected exclusively in the epidermal cell layer, and transgene expression of IL-12 cDNA was maximal 2 days post-transfection and remained detectable for at least 5 additional days. Furthermore, particle-mediated delivery of IL-12 cDNA into epidermal cells overlying an intradermal tumor resulted in a significant suppression of tumor growth of Lewis lung carcinoma. Appreciable levels of IFN-gamma production were readily detected at the skin transfection site, and were induced from splenocytes and lymph node cells in the IL-12 treated mice. These results show that in vivo delivery of IL-12 cDNA into skin by the Helios gene gun device can have a useful routine application for cancer therapy research.     

Effects of perchlorate on sodium-iodide symporter and pendrin gene expression in deer mice

Effects of perchlorate on sodium-iodide symporter (NIS) and pendrin gene expression in deer mice kidney and stomach were investigated. This was accomplished by isolating a partial cDNA sequence of deer mice NIS gene of 425 bps, and quantitatively analyzing NIS mRNA expression in various deer mouse tissues. The highest NIS expression level was in the stomach, followed by testes, brain, and large intestine; very low expression of NIS was observed in the lung, kidney, heart, and liver. Exposure to perchlorate through drinking water for 28 days did not significantly increase NIS gene expression in the kidney and stomach, and pendrin gene expression in the kidney. In a depuration experiment in which deer mice were exposed to perchlorate for 8-h followed by an 88-h depuration period, no significant difference was observed between the low and high exposure groups in terms of NIS or pendrin gene expression in the kidney or stomach at the end of the experiment. Furthermore, no significant linear relationship was observed between gene expression (either NIS or pendrin) in the kidney and perchlorate mass excreted via urine at day 28, average daily excretion, or total excretion mass over the 28 day exposure. Several factors could influence the effect of perchlorate exposure on NIS and pendrin gene expression in the stomach and kidney, including (1) pre-exposure to trace perchlorate through food and water perhaps resulting in adaptation (or tolerance) in these animals; (2) metabolism of perchlorate in deer mice causing only 46-61% perchlorate excreted into urine. It is also possible that there is no effect of perchlorate exposure and/or urinary excretion on NIS and pendrin gene expression, particularly in the kidney.     

牛气管抗菌肽cDNA的克隆与表达

目的：开发一种有效抑制动物细菌感染的重组抗菌肽。方法：根据已发表的牛气管抗菌肽（bTAP）mRNA序列设计引物，用RT—PCR从奶牛气管上皮细胞总RNA中扩增出214bp的bTAPcDNA，将其克隆入pUCm—T载体进行序列测定，将此cDNA分别克隆入真核表达载体pcDNA3和原核表达载体pGEX-6p-1进行表达研究。结果：测序结果与已发表的bTAP序列有3个碱基差异，其中2个导致氨基酸替换；经微球菌溶解试验证明，注射在小鼠皮下的重组质粒pcDNA—tap能表达有活性的bTAP；抗体测定结果显示重组质粒pcDNA—tap经5次免疫后家兔产生的抗bTAP抗体效价达1：800；经IPTG诱导后，含重组质粒pGEX—tap大肠杆菌能表达预计大小的GST—bTAP融合蛋白，且能被兔抗bTAP识别。结论：本研究克隆的bTAPcDNA可用于表达研究及相关基因工程产品的开发。