Transfected Cell Clones Research Articles

Long-Term Survival of Cellulose Sulphate-Encapsulated Cells and Metronomic Ifosfamide Control Tumour Growth in Pancreatic Cancer Models-A Prelude to Treating Solid Tumours Effectively in Pets and Humans.

The use of encapsulated cells for the in vivo delivery of biotherapeutics is a promising new technology to potentiate the effectiveness of cell-based therapies for veterinary and human application. One use of the technology is to locally activate chemotherapeutics to their short-lived highly active forms. We have previously shown that a stable clone of HEK293 cells overexpressing a cytochrome P450 enzyme that has been encapsulated in immunoprotective cellulose sulphate beads can be implanted near solid tumours in order to activate oxazaphosphorines such as ifosfamide and cyclophosphamide to the tumour-killing metabolite phosphoramide mustard. The efficacy of this approach has been shown in animal models as well as in human and canine clinical trials. In these previous studies, the oxazaphosphorine was only given twice. An analysis of the Kaplan-Meier plots of the results of the clinical trials suggest that repeated dosing might result in a significant clinical benefit. In this study, we aimed to (i) demonstrate the stable long-term expression of cytochrome P450 from a characterized, transfected cell clone, as well as (ii) demonstrate that one implanted dose of these encapsulated cytochrome P450-expressing cells is capable of activating multiple doses of ifosfamide in animal models. We initially used cell and molecular methods to show cell line stability over multiple passages, as well as chemical and biological function in vitro. This was followed by a demonstration that encapsulated HEK293 cells are capable of activating multiple doses of ifosfamide in a mouse model of pancreatic cancer without being killed by the chemotherapeutic. A single injection of encapsulated HEK293 cells followed by multiple rounds of ifosfamide administration results in repeated anti-tumour activity and halts tumour growth but, in the absence of a functioning immune system, does not cause tumour regression.

Assessing the allogenic realness of the Cw1/12/15 pattern occurring in the LABScreen single antigen assay.

Several technical limitations of Luminex single antigen (LSA) assays have been described so far. This study focused on a reactivity pattern observed in many sera that cannot be explained by eplets described in the Epitope Registry database and sometimes appearing against a self-HLA allele or antigen. In most cases, this pattern is revealed by a discrepant result when compared with other assays (Luminex PRA, cell-binding assays such as flow cytometry cross match, LSA from another manufacturer…). We focus here on the Cw1/12/15 pattern appearing on the LABScreen class I LSA provided by One Lambda. We documented its behavior using this LSA after acid denaturation of the beads, using Lifecodes LSA from Immucor, and adsorption of sera either on spleen mononuclear cells from deceased donors or on single HLA transfected cell clones. We studied 33 sera from different patients positive for the three Cw beads, selected from our routine patients' LSA database. Nine patients had transplants from a Cw12 or Cw15 donor without any pejorative evolution of the graft, nor post-transplant MFI (mean fluorescence intensity) increase of the Cw1/12/15 beads. A significant increase of MFI was observed after acid denaturation of the LABScreen beads. All sera tested by Lifecodes LSA were negative for these Cw beads. Finally, we found no significant difference of MFI after adsorption on cells from either origin. Therefore, the Cw1/12/15 pattern appears to be a false positive reactivity of the LABScreen single antigen assay.

Inducible Expression of Amphinase in Neuroblastoma Cells using Cre/LoxP System

PurposeTo induce the expression of Amphinase, an antitumor ribonuclease from Rana pipiens oocytes, in neuroblastoma cell lines and build a foundation for mechanism study. MethodsA loxP-cassette vector was constructed comprising a sequence of loxP -Puro-3∗polyA-loxP, followed by amphinase cDNA. The vector was transfected into neuroblastoma cell lines, SK-N-BE(2)-C, by Lipofectamine LTX. The transfected cells were selected by puromycin for two weeks. Polymerase chain reaction (PCR) and real-time quantitative PCR (qPCR) were conducted to verify that the loxP-cassette vector was stably transfected. The expression of amphinase was activated by the addition of Cre recombinase delivered by a lentiviral vector and identified by qPCR and Western blotting (WB). CCK8 assay and colony formation assay were conducted to check the effect of amphinase on cell proliferation. RNA sequencing (RNA-seq) was conducted to explore the targeted pathway of Cre/loxP-mediated amphinase and recombinant amphinase. ResultsStably transfected cell clones were achieved through puromycin selection. After Cre recombinase was delivered to the cells, the loxP-flanked fragment was deleted and the expression of amphinase was induced, which were tested by PCR and qPCR. It was shown that cell proliferation was significantly inhibited by the amphinase mediated by the Cre/loxP system. KEGG enrichment and GSEA analysis indicated that amphinase had an impact on the ER function of neuroblastoma cells, which was identical to the effect of the recombinant amphinase. ConclusionWe successfully induce the expression of amphinase in neuroblastoma cell lines via Cre/loxP system. The Cre/loxP-mediated amphinase had a similar antitumor mechanism to the recombinant amphinase, providing a powerful tool for the mechanism study of amphinase.

Japanese encephalitis genotype I virus-like particles stably expressed in BHK-21 cells serves as potential antigen in JE IgM ELISA.

Japanese encephalitis virus (JEV) is one of the leading causes of epidemic encephalitis in South Asian countries. Due to the short-term viremia, detecting IgM antibodies by ELISA is treated as the front-line diagnostic assay. Co-circulation and multiple exposures to antigenically cross-reactive flaviviruses in India pose a challenge in serodiagnosis. Replacing the whole virus antigen currently used in the JE IgM detection kits (ELISA) may improve the specificity and sensitivity of the existing JE MAC ELISA kits. For this purpose, we developed a stably transfected cell clone, BHK-IE6, which expresses a high amount of VLPs up to 37 µg/ml and is consistent in expression up to 40 passages. For the expression of VLPs in the secretory form, we cloned the JEV G-I prM-E coding gene along with the C-terminal signal sequence of capsid protein in the BHK-21 cells using the pcDNA3.1 + mammalian expression vector. The immune assays performed demonstrated its immune reactivity equivalent to the parental JEV strain. Simultaneously performed ELISAs using the whole virus antigen and newly developed antigen gave comparable results for JE positive and negative samples, which established the utility of developed JEV E-VLP as an antigen. Reduced cross-reactivity and increased specificity were observed when tested with dual positive sera for anti-JEV and DENV antibodies. These findings confirm the efficiency and reliability of newly developed recombinant E-VLP antigen expressed by the BHK-IE6 cell clone as an antigen in serodiagnostic assays. The implementation and progress in developing cross-reactivity-reduced antigens would improve serodiagnosis and disease burden estimates of flavivirus infection.Key points • pcDNA3.1/JE-Sig-prM-E plasmid transfected BHK-21 cells stably express VLPs. • Sodium butyrate induction enhanced the extracellular expression of VLPs. • Application of JEV-E VLPs increases the specificity of JE IgM ELISA. Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-022-11825-1.

14-3-3ζ protein mediates gemcitabine resistance in NK/T-cell lymphoma

目的探讨14-3-3ζ蛋白在结外NK/T细胞淋巴瘤，鼻型（ENKTL）吉西他滨耐药中的分子机制。方法CCK-8法检测细胞增殖，Transwell小室法检测细胞的侵袭性，逐步增加吉西他滨浓度建立吉西他滨耐药YTS细胞系（YTS-gem），MTT法检测药物敏感性，Western blot法检测14-3-3ζ蛋白在YTS-gem细胞和YTS细胞中的表达差异。采用siRNA下调14-3-3ζ表达，对比下调前后YTS-gem细胞对吉西他滨敏感性的差异。Western blot法检测耐药相关蛋白在14-3-3ζ下调前后YTS-gem细胞中的表达差异。结果①与YTS细胞相比，14-3-3ζ在YTS-gem细胞中表达上调，差异有统计学意义（P<0.05）；②与对照组相比，下调14-3-3ζ后YTS细胞和YTS-gem细胞的增殖和侵袭能力均明显受抑（P<0.05）；③下调14-3-3ζ表达后，YTS-gem细胞对吉西他滨敏感性增加，差异有统计学意义（P<0.05）；④下调14-3-3ζ表达后，YTS-gem细胞中促凋亡蛋白Bax水平显著升高，抗凋亡蛋白Bcl-2、caspase-3、cleaved caspase-3和Cyclin D1显著降低（P<0.05），而P-gp和p53表达水平的差异无统计学意义。结论14-3-3ζ在YTS-gem细胞中表达上调，14-3-3ζ促进细胞增殖和侵袭，14-3-3ζ蛋白通过抗凋亡途径诱导ENKTL对吉西他滨耐药。

Effect of Inhibiting NCL Expression on Drug-Resistance of Chronic Myeloid Leukemia Cell Line K562 and Its Resistant Cells

To construct a K562 and adriamycin-resistant K562 (KAR) cell line with stably down-regulation of NCL (nucleolin) expression, and to investigate the effect of NCL down-regulation on the drug resistance in K562 and KAR cells. K562 and KAR cells were infected with lentivirus, and stably transfected cell clones were obtained by puromycin screening. The cell proliferation was detected by MTS assay, the cell apoptosis was detected by flow cytometry, and the expression level of drug resistance related genes was detected by real-time PCR. The K562 and KAR cells with stable down-regulation of NCL were successfully constructed. Compared with the control group, the proliferation of K562 and KAR cells with down-regulating NCL expression decreased significantly (P ＜0.05), the apoptosis of cells increased significantly (P ＜0.05), and cell resistance to adriamycin was down-regulated. Inhibition of NCL expression may increase the sensitivity of cells to adriamycin, which may be related with the promotion of apoptosis of K562 and KAR cells.

The effects of up-regulation of nuclear Clusterin gene on the biological behaviors of A549 cells

Objective:To observe the up-regulation of nuclear Clusterin (nCLU)gene on the biological behaviors of human non-small cell lung cancer cell line A549.Methods:Sense eukaryotic expression vector of nCLU was constructed by cloning the cDNA of nCLU into pIREShyg3 vector. A549 cells were transfected with pIRES-nCLU and pIREShyg3 vectors by lipofectAMINETM 2000 mediation,respectively. Stable transfected cells were selected by hygromycin B screening. CCK-8 assay was performed to evaluate the effect of nCLU over-expression on cell proliferation in vitro. The expression level of nGLU protein was examined by Western blotting. Cell cycle distribution was detected by FCM with PI staining. The alteration of migration and metastasis potential of A549 cells before and after nCLU gene transfection were assayed by cell chemotactic migration and invasion test. Results:The proliferation speed of the transfected A549 cell clones stably over-expressing nCLU was slowed down. FCM analyses revealed that the percentage of cells in G0/G1 phase dramatically increased from (33.54±2.10)% to (63.31±4.30)%. The cell chemotactic migration and invasion potentials were markedly reduced after nCLU gene transfection (P0.05). Conclusion:Up-regulation of nCLU can greatly inhibited the proliferation and decreased the migration and invasion capabilities of A549 cells.

Effective combination of xenoprotective transgenes

Abundant expression of a series of xenoprotective transgenes is vital for clinically useful xeno‐donor pigs. Breeding of such multi‐transgenic animals requires that xeno‐transgenes be placed at a single, or a small number of loci in the pig genome to avoid or minimise separation by genetic segregation. We are investigating methods that support transgene expression and also enable transgenes to be “stacked” at a single locus. These include the use of BAC based multi‐transgene vectors, and very recently serial transgene placement at a permissive locus by homologous recombination supported by TALENs [1].BAC vector constructs containing genomic sequences for the complement activation inhibitors CD55, CD46 and CD59 were assessed to determine the minimum size without reduction of expression level. These constructs enable us to express all biologically active CD55 splice variants (membrane bound and soluble forms). Effective xenoprotection also requires ubiquitous expression. We are thus examining endogenous promoter sequences of various lengths as well as the widely used CAGGs (CMV enhancer/chicken β actin) promoter to direct complement inhibitory gene expression levels in all porcine tissues.To investigate means of coexpressing groups of transgenes that provide xeno‐protection at different levels, we developed a series of BAC vector constructs containing the CD55 genomic sequence plus two additional xeno‐transgene cDNAs, including A20 [2], HO1 [3], thrombomodulin [4] and CTLA4‐Ig (LEA29Y) [5].Analysis of porcine adipose MSC stably transfected cell clones revealed expression of all inserted xeno‐transgenes most importantly high expression of complement inhibitory genes (Fig. 1). This demonstrates that xeno‐transgenes can be effectively combined in a single construct, thus minimising the number of transgene loci in the final donor pigs. The huge carrying capacity of BAC vectors enables the addition of further xeno‐transgenes in a single vector construct.We have further examined the system in vivo. Primary cells transfected with up to three BAC constructs were used for nuclear transfer, fetuses explanted and examined for expression with very promising results.

Determination of Transgene Copy Number in Stably Transfected Mammalian Cells by PCR-Capillary Electrophoresis Assay

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.

Milligram Production and Biological Activity Characterization of the Human Chemokine Receptor CCR3

Human chemokine receptor CCR3 (hCCR3) belongs to the G protein-coupled receptors (GPCRs) superfamily of membrane proteins and plays major roles in allergic diseases and angiogenesis. In order to study the structural and functional mechanism of hCCR3, it is essential to produce pure protein with biological functions on a milligram scale. Here we report the expression of hCCR3 gene in a tetracycline-inducible stable mammalian cell line. A cell clone with high hCCR3 expression was selected from 46 stably transfected cell clones and from this cell line pure hCCR3 on a milligram scale was obtained after two-step purification. Circular dichroism spectrum with a characteristic shape and magnitude for α-helix indicated proper folding of hCCR3 after purification. The biological activity of purified hCCR3 was verified by its high binding affinity with its endogenous ligands CCL11 and CCL24, with K D in the range of 10−8 M to 10−6 M.

325 PRODUCTION OF GAL KNOCKOUT/hA20 TRANSGENIC PIGS WITH IMPROVED XENOPROTECTIVE PROPERTIES

Pig-to-human xenotransplantation is promising for overcoming the shortage of suitable human donor organs, but is hampered by immunological barriers. The next immunological hurdle is the acute vascular rejection (AVR), which is associated with activation of the endothelium and the coagulation system. Recently, we demonstrated that transgenic expression of the zinc finger protein A20 protects porcine cells against apoptotic and inflammatory stimuli (Oropeza et al. 2009 Xenotransplantation 16, 522–534). Compared with other anti-apoptotic proteins, A20 also has immune-modulatory potential as shown in an CD95(Fas)Ligand assay. However, in that study, hA20 was only expressed in skeletal muscle, heart and porcine aortic endothelial cells of transgenic pigs. For use in xenotransplantation, it is critical to produce pigs with ubiquitous expression of hA20. Here, we constructed a new vector based on the Sleeping Beauty transposon plasmid pT2/HB containing the hA20 cDNA driven by the ubiquitously and strongly expressing CAGGS-promoter and co-transfected gal–/– porcine fibroblasts (Hauschild et al. 2011 Proc. Natl. Acad. Sci. USA 108, 15 010) together with the SB transposase 100X plasmid (pT2/HB and SB transposase both kindly provided by Dr. Zoltan Ivics). Cells were selected with 400 µg of G418/mL of medium for 14 days. Subsequently, cells were screened by PCR. Transfected cell clones were pooled and used as donor cells in somatic cell nuclear transfer. Reconstructed embryos were transferred to 2 synchronized sows. Both remained pregnant on Day 25 of gestation. One recipient is expected to deliver in September 2012. The second sow, which received 104 embryos, was sacrificed on Day 26 of pregnancy and 2 fetuses could be obtained (cloning efficiency 1.92%). The fetuses had integrated the transgene into their genome as shown by PCR. Real-time PCR results from fetal fibroblasts indicated similar hA20 mRNA expression levels in both fetuses, whereas wild type controls were negative. The hA20 expression level was 2.5 and 3.1 times lower than in the original pooled of transfected cells. Fetus #1, which showed a slightly higher hA20 mRNA expression, was used for recloning. In total, 3 more recipients received an average of 104 hA20 transgenic embryos each. Currently, the hA20 protein level in fetal fibroblasts is determined by fluorescence activated cell sorting analysis. Once pigs are born, the tissue distribution of hA20 will be analysed. In parallel, the function of the transgene will be studied in the CD95(Fas)Ligand assay. Hearts and kidneys of the hA20-transgenic pigs will be further tested in ex vivo perfusion assays and in a pig-to-baboon xenotransplantation. This approach is promising for advancing pig-to-human xenotransplantation to preclinical application.

Induction of autoantibodies against mouse soluble proteins after immunization with living cells presenting the autoantigen at the cell surface in fusion with a human type 2 transmembrane protein

Induction of autoantibodies towards immune regulatory proteins, such as cytokines or their receptors, is a powerful strategy for functional studies on the role of these factors in vivo. Here we describe a new procedure to elicit autoantibodies by taking advantage of tumor cells as a vaccine against peptides presented at their surface in fusion with the human CD134L transmembrane protein. P1.HTR, an immunogenic variant of the P815 mastocytoma cell line, was used to generate stably transfected cell clones with expression vectors encoding the human CD134L transmembrane protein fused with either mouse IL-22BP or IL-9. Following repeated injections of living tumor cells expressing the mIL-22BP construct, mice developed autoantibodies that bind to mIL-22BP and inhibit its interaction with IL-22 in vitro. Mice similarly immunized against mIL-9 produced high titers of autoantibodies that block the activity of this cytokine in the TS1 bioassay. This procedure also inhibits IL-9 activity in vivo as no increase of serum MMCP-1 mast cell protease concentration was observed following IL-9 administration to immunized mice. As an alternative to the injection of living tumor cells expressing the CD134L-antigen fusion protein, intramuscular electrotransfer of the corresponding DNA construct also induced autoantibodies. These results validate this method as a simple and convenient approach to knock down the in vivo activity of soluble regulatory proteins, including cytokines and their receptors.

New transgenic pigs for xenotransplantation, part 2: Strategies to overcome cellular rejection

The successful generation of α-1,3 galactosyltransferase knockout (αGal-KO) pigs and pigs expressing human complement regulators was a crucial step forward in xenotransplantation research. These genetic modifications helped to control hyperacute rejection, but vascular and cellular mechanisms still represent a major hurdle for pig-to-primate xenotransplantation. Coagulation incompatibilities between human blood and porcine endothelium lead to thrombotic microangiopathy, which could be observed as a key rejection mechanism in pig-to-primate transplantation using αGal-KO pigs as organ donors. Circulating human thrombin is bound by porcine thrombomodulin, but the resulting heterodimer fails to activate protein C, which inhibits the coagulation cascade. We established a vector for endothelial-specific expression of human thrombomodulin (hTM) in αGal-KOxCD46 pigs. The hTM construct was combined with a floxed blasticidin resistance cassette and transfected mixed cell populations were used as donors for somatic cell nuclear transfer (SCNT). Two hundred and ninety-eight reconstructed embryos were transferred to four recipients, resulting in one pregnancy, which lead to the birth of three living piglets. All of them showed strong endothelial hTM expression. In contrast to vascularised organs, pancreatic islets are not subject to hyperacute rejection, but to cellular rejection, mainly based on T-cell stimulation as islets of mature pigs express αGal-epitopes only at a very low level and are vascularized by recipient capillaries after transplantation. Human CTLA4-Ig is a potent inhibitor of T-cell activation, excelled by its derivate LEA29Y which has higher binding affinity to the costimulatory B7 receptors. We have established the coding sequence of LEA29Y under the transcriptional control of a regulatory fragment of the porcine insulin gene. The construct also contained a floxed neomycin resistance cassette. Porcine fetal fibroblasts were transfected, followed by selection under geneticin for 7 days. Stably transfected cell clones were pooled and used as donor cells for SCNT. Transfer of 216 reconstructed embryos to three recipients resulted in two pregnancies with nine born piglets, including two stillbirths. Seven piglets were shown to be transgenic. Immunohistochemistry of the pancreata of two transgenic founders showed high expression levels of LEA29Y at an age of 3 months.

Transgenic strategies to overcome cell‐mediated and acute vascular rejection of pig‐to‐human xenografts

The development of α(1,3)‐galactosyltransferase deficient pigs along with overexpression of complement regulatory proteins was an important step to overcome hyperacute rejection of pig organs transplanted into primates. However, the lack of galactose‐α(1,3)‐galactose (αGal) epitopes does not protect against subsequent rejection mechanisms, such as acute vascular rejection and rejection by immune cells which play crucial roles to achieve prolonged graft survival in pig‐to‐human xenotransplantation models. Our task within the DFG Transregio Research Unit “Xenotransplantation” is primarily the development of transgenic strategies to overcome cell mediated rejection of pig‐to‐primate xenografts. Our first target was the lysis of porcine cells by activated human natural killer (NK) cells, which involves antibody‐dependent and ‐independent mechanisms. A majority of human NK cells express the inhibitory receptor CD94/NKG2A, which binds specifically HLA‐E, a trimeric complex consisting of the HLA‐E heavy chain, β2‐microglobulin (β2m) and a peptide derived from the leader sequence of some MHC class I molecules. To use this mechanism for protection of pig tissues against human NK cell‐mediated cytotoxicity, we generated transgenic pigs carrying genomic fragments of HLA‐E with a HLA‐B7 signal sequence and of human (hu) β2m. Immunohistochemistry revealed the presence of HLA‐E and huβ2m on endothelial cells of many organs, including heart and kidney. In vitro studies showed that lymphoblasts and endothelial cells derived from HLA‐E/huβ2m transgenic pigs are effectively protected against human NK cell‐mediated cytotoxicity, depending on the level of CD94/NKG2A expression on the NK cells. Further, HLA‐E/huβ2m expression on porcine endothelial cells inhibited the secretion of IFN‐γ by co‐cultured human NK cells [1]. Currently the HLA‐E/huβ2m transgenes are crossed on a CD46 transgenic background to facilitate evaluation of the NK cell inhibitory effect in ex vivo perfusion studies of multitransgenic pig hearts with human blood.The second target is inhibition of T cell activation by expression of CTLA‐4Ig. This recombinant soluble fusion protein binds to CD80 and CD86, blocking their interaction with CD28 and thereby inhibiting T cell‐dependent antibody production and T cell proliferation. The survival of human, rabbit and porcine islets after transplantation into diabetic mice was found to be prolonged after treatment with CTLA‐4Ig. As a transgenic strategy to protect porcine islets against human T cells, we cloned an expression vector, in which the coding sequence for LEA29Y, a more potent variant of CTLA‐4Ig, is placed under the transcriptional control of the porcine INS promoter. This expression cassette is linked to a floxed neomycin resistance cassette in order to facilitate selection of stably transfected fetal fibroblasts (FF). After two passages under selection, pooled FF colonies were used for nuclear transfer. Cloned embryos were laparoscopically transferred to recipient gilts, resulting in two pregnancies and eight offspring, seven of which were transgenic. Immunohistochemistry of pancreas sections from one selected founder demonstrated the presence of LEA29Y in the pancreatic islets, the expression pattern being in agreement with β‐cell specific expression. Protection of INS‐LEA29Y transgenic islets will be tested in diabetic mouse models with a humanized immune system.In addition to cell mediated rejection mechanisms we attempted to target acute vascular rejection mechanisms by expression of human thrombomodulin (hTM) on porcine endothelial cells. This has been discussed as an important step to overcome incompatibilities in the regulation of human blood coagulation after perfusion of porcine blood vessels. To achieve efficient expression of hTM we constructed three different expression vectors: (i) the hTM gene with homologous 5′ sequences and polyadenylation signal; (ii) the hTM gene driven by the porcine TM promoter, but with homologous 3′ flanking sequences; and (iii) the hTM gene with the porcine TM promoter and a polyadenylation signal and 3′ flanking sequences from the bovine growth hormone gene. The latter expression vector was linked to the neomycin resistance cassette mentioned above and transfected into fetal fibroblasts. A pool of stably transfected cell clones was used for nuclear transfer. Transfer of cloned embryos to recipients resulted in three pregnancies and eight piglets, all of which were transgenic. Five of six founders investigated showed strong expression of hTM on endothelial cells in heart and kidney. Cells from selected animals are currently being used for re‐cloning to provide transgenic pig organs for ex vivo perfusion studies with human blood to evaluate the efficacy of hTM expression.Supported by the DFG (FOR 535 “Xenotransplantation”).Reference1. Weiss, et al. HLA‐E/human beta2‐microglobulin transgenic pigs: protection against xenogeneic human anti‐pig natural killer cell cytotoxicity. Transplantation 2009; 87: 35–43.

A versatile nonviral vector system for tetracycline-dependent one-step conditional induction of transgene expression

In this study, we describe a novel self-contained, nonviral vector system for the rapid development of tetracycline (Tet)-inducible transgene expression systems in mammalian cell lines. To avoid multiple rounds of clonal selection for the establishment of stably transfected cell clones, as is necessary with conventional systems, we constructed a multicomplementary DNA(cDNA) expression vector that enables both one-step targeted genomic integration and conditional induction of transgene expression. This vector system consists of several modules including a Tet-inducible promoter directing the expression of a transgene and two Tet repressor expression units placed in tandem on a single vector. The cell clones, generated using a one-step phiC31 integrase-mediated chromosomal integration of the multi-cDNA expression construct, showed a stable and robust expression with high induction rates upon addition of doxycycline inducer in five different cell lines tested. By using this system, we show c-Src-induced cell transformation and anticancer cell therapy for this transformation in cultured fibroblast cells. The results show a rapid production and accumulation of target protein on addition of the inducer starting from extremely low background levels and reduction to background levels in a matter of days after the inducer was withdrawn from the culture medium.

Role of short haipin RNA targeting vascular endothelial growth factor-C on biological characteristics of human breast cancer cell MCF-7

To study the effect of short haipin RNA (shRNA) on expression of vascular endothelial growth factor C (VEGF-C) and proliferation and invasion behavior of human breast cancer cell MCF-7. The recombinant vector (pSIREN-VEGF-C) was transfected into the human breast cancer cell MCF-7 by liposome and the positive transfected cell clones were screened with puromycin. Expression of VEGF-C in MCF-7 cells after gene transfer was detected by real-time quantitative PCR and Western blot assay, respectively. Proliferation and invasion ability of transfected cells were analyzed by MTT and Transwell filter. The expressions of VEGF-C mRNA and protein were decreased markedly compared with the control group after the transfection and the inhibitive ratio was 95% and 100% respectively (P<0.05). The proliferation of MCF-7 cells transfected by pSIREN-VEGF-C, measured with MTT assays, was significantly decended (P<0.05). The invasion ability of passing through the Transwell filter of MCF-7 cells transfected by pSIREN-VEGF-C were declined evidently (P<0.05). The recombinant vector (pSIREN-VEGF-C) have been proved not only to be effective and specific for down-regulation of VEGF-C, but also can inhibit the proliferation and invasion of MCF-7 cells significantly.

Effect of Fluorouracil-inducible GM-CSF gene therapy regulated by Egr-1 promoter on chemotherapeutic hematopoietic damage of tumor-bearing mice

In order to explore the regulating effects of Egr-1 promoter sequences in transcriptional targeting by 5-fluorouracil (5-Fu) on the expression of hematopoietic growth factor genes. The human GM-CSF cDNA and enhanced green fluorescent protein (EGFP) cDNA were linked together with IRES and then inserted into the expression vector pCIneo under control of the Egr-1 promoter (Egr-EG). The vector was transferred into human bone marrow stromal cell line HFCL by lipofectin(TM). The transfected cell clones (HFCL/EG) have been selected by the addition of G418. The cells are exposed to the clinically important anticancer agent 5-fluorouracil. The activity of EGFP in HFCL/EG cells was detected by flow cytometry. The post-chemotherapeutical expression of GM-CSF in HFCL/EG was confirmed with ELISA and Western blot and RT-PCR respectively. The effect of N-acetylcysteine (a free radical scavenger) on GM-CSF production post-exposure to 5-Fu was examined. The HFCL/EG cells were transplanted intravenously into B16 melanoma in C. B-17 combined immunodeficient (SCID) mice. 5-Fu was given i.p. at Day 3. The white blood cell number in peripheral blood, the expression of EGFP and GM-CSF and in human stromal cell engrafted in recipient mice were detected by flow cytometry and RT-PCR respectively. Tumor volume in tumor-bearing mice was calculated. The results indicated that the activity of EGFP and the amounts of secreted GM-CSF in HFCL/EG cells exposed to 5-Fu increased as compared to non-5-Fu group with flow cytometry, RT-PCR and ELISA respectively. N-acetylcysteine significantly decreased the concentration of GM-CSF in HFCL/EG cells treated with 5-FU. In contrast to two control groups, HFCL/EG (Egr-1 regulatory element-derived expression of GM-CSF gene therapy) resulted in a proportionally obvious increase in the number of white blood cell after chemotherapy and no significant difference was found for tumor inhibition in recipient mice. These in vitro data provide an experimental basis for use of gene therapy of hematopoietic growth factor gene regulated by Egr-1 promoter to protect hematopoiesis from 5-Fu-injury effects.

Antisense expression of PKCalpha improved sensitivity of SGC7901/VCR cells to doxorubicin

To explore whether antisense blocking of protein kinase C alpha (PKCalpha) would reverse multi-drug resistance (MDR) in the vincristine (VCR)-resistant human gastric cancer cell line SGC7901/VCR. SGC7901/VCR cells expressing antisense PKCalpha, SGC7901/VCR/aPKC, were established by transfection with a recombinant plasmid reversely inserted with PKCalpha cDNA. Empty vector (PCI-neo)-transfected cell clones, SGC7901/VCR/neo, served as the control. Western blot method was used to detect PKCalpha content in SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells, using PKCalpha-specific antibody. The sensitivity of SGC7901, SGC7901/VCR, SGC7901/VCR/neo and SGC7901/VCR/aPKC cells to doxorubicin (DOX) in vitro was determined by MTT assay. The uptake of DOX in these cells was detected with fluorescence spectrophotometer. Western blot analysis showed that the PKCalpha protein level was about 8.7-fold higher in SGC7901/VCR cells than that in SGC7901 cells, whereas the protein expression of PKCalpha was reduced by 78% in SGC7901/VCR/aPKC cells when compared with the SGC7901/VCR cells. SGC7901/VCR/aPKC cells had a 4.2-fold increase in DOX cytotoxicity, accompanied by a 1.7-fold increase of DOX accumulation in comparison with SGC7901/VCR cells. PKCalpha positively regulates MDR in SGC7901 cells, and inhibition of PKCalpha can partially attenuate MDR in human gastric cancer cells.

Influence of Skp2-targeted siRNA on characteristics of human colorectal cancer cell line

AIM:To design and construct the RNAi eukaryotic vector of Skp2 gene and to study its interfering effect on characteristics of human colorectal carcinoma cell line HCT116.METHODS:DNA oligonucleotide for small interfering RNA expression targeting Skp2 gene was synthesized and inserted into pRNAT-U6.1/Neo plasmid after annealing. The inserted se-quences were verified by DNA sequencing,and transfected into HCT116 cells with liposome-mediated transfection. The positive transfected cell clones were screened with G418 and the ex-pression of Skp2 mRNA was detected by semi-quantitive RT-PCR. Distribution of cell cycle was assessed by flow cytometry. The cell growth suppression was analyzed by MTT assay. RESULTS:The sequence of specific siRNA wascorrect by sequence analysis. Compared with the negative control cells,RT-PCR showed the expression of Skp2 mRNA was down-regulated in the transfected cells (P 0.05). The subcloned recombinant plasmid expressing shRNA effec-tively inhibited HCT116 cell growth and prolif-eration while empty plasmid had no such specific effect. Decreased cellular proliferation activity was observed by MTT (29.21% ± 1.40%,30.10% ± 1.50% vs 49.05% ± 4.50%,53.03% ± 4.92%,all P 0.05). Flow cytometry revealed signifi cant G1 ar-rest in cell cycle. CONCLUSION:The siRNA eukaryotic expression vector of Skp2 gene was constructed suc-cessfully which effectively down-regulate the ex-pression of Skp2 in HCT116 cell line,and inhibit the cellular proliferation. It provides a powerful evidence for colorectal carcinoma gene therapy.

Differential role of α vβ 3 and α vβ 5 integrins in internalization and transduction efficacies of wild type and RGD4C fiber-modified adenoviruses

In order to analyze the role of α vβ 3 and α vβ 5 integrins in gene transfer by adenovirus-based vectors, an RGD4C motif was inserted into the HI-loop of wild type or shortened fiber protein of human adenovirus of serotype 5, thereby creating Ad5RGD4C and Ad5Δ639RGD4C vectors, respectively. Infection by the latter is independent of the Coxsackie B adenovirus receptor. Internalization and transduction of these vectors and were investigated in several stably transfected cell clones derived from a human laryngeal carcinoma cell line (HEp2) expressing different ratios of α vβ 5 and α vβ 3 integrins. We show that α vβ 3 is more successful than α vβ 5 in: (i) mediating adenovirus internalization and transduction when the RGD motif is present only in the penton base and (ii) mediating internalization and transduction by RGD4C-fiber modified adenoviruses. The highest amount of internalized virus was found in the cell clone in which α vβ 3 integrin predominated over α vβ 5 integrin (as judged by the % of cells expressing α vβ 3 and α vβ 5 integrins). However the level of transgene expression in this cell line was even lower than that in parental HEp2 cells which do not express α vβ 3 integrin. This discrepancy between internalization and transgene expression (transduction) is likely due to the crucial role of α vβ 5 in membrane permeabilization, indicating that α vβ 5 integrin is a limiting factor for Ad5-mediated gene transfer. We conclude that α vβ 3 integrin is an efficient adenovirus internalization receptor, but cannot functionally replace α vβ 5 in endosomal release.