Expression of recombination-activating genes and T cell receptor gene recombination in the human T cell leukemia cell line

Background Recent studies have suggested that mature T cells can change their specificity through reexpression of recombination-activating genes （RAG） and RAG-mediated V（D）J recombination. This process is named receptor revision and has been observed in mature peripheral T cells from transgenic mice and human donors. However, whether the receptor revision in mature T cells is a random or orientated process remains poorly understood. Here we used the Jurkat human T cell line, which represents a mature stage of T cell development, as a model to investigate the regulation of T cell receptor （TCR） gene recombination. Methods TCR Dβ-Jβ signal joint T cell receptor excision DNA circles （sjTRECs） were determined by nested and seminested PCR. Double-strand DNA breaks at recombination signal sequences （RSSs） in the TCRVβ chain locus were detected by ligation-mediated-PCR. Further analysis of the complementarity-determining region 3 （CDR3） size of the TCRVβ chain was examined by the TCR GeneScan technique. Results RAG1, RAG2, and three crucial components of the nonhomologous DNA end-joining （NHEJ） pathway were readily detected in Jurkat. Characteristics of junctional diversity of Dβ2-Jβ2 signal joints and ds RSS breaks associated with the Dβ2 5＇ and Dβ 2 3＇ sites were detected in DNA from Jurkat cells. CDR3 size and the gene sequences of the TCRVβ chain did not change during cell proliferation. Conclusions RAG1 and RAG2 and ongoing TCR gene recombination are coexpressed in Jurkat cells, but the ongoing recombination process may not play a role in modification of the TCR repertoire.However, the results suggest that Jurkat could be used as a model for studying the regulation of RAGs and V（D）J recombination and as a ＂special＂ model of the coexistence of TCR gene rearrangements and ＂negative＂ receptor revision.

Genetic Analysis of the Entire Genome of a A/duck/Shanghai/Y20/2006 (H4N6) Avian Influenza Virus

[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl origin AIV. [Method] The entire genome of DK/SH/Y20/06 was amplified and subjected to genome sequencing. The molecular software was used for sequence analysis and phylogenetic tree construction of DK/ SH/Y20/06 with some other reference sequences in GenBank. [Result] The results indicated that the amino acid sequence adjacent to HA cleav- age site was PEKASR ↓ GLF, which was the typical characteristics of the LPAIV. The phylogenetic analysis indicated that the HA gene of the isolate was derived from the Eurasian lineage in the eastern hemisphere. The NA gene was at the same branch with A/rnallard/Yan chen/2005（ H4N6）, sharing 98.3% sequence identity. The PB2, PB1, NP and PA gene of this isolate had genetically close relationships with H6 subtype AIV which is epidemic in China at present. The M gene fell into the same branch with A/environment/Korea/CSM05/2004（ H3N1 ）. The NS segment had the highest similarity with A/wild duck/Korea/YS44/2004（H1N2）. The eight genes were not at the same branch and shared a low similarity with other H4N6 subtype avian influenza viruses isolated in North America. [Condusion] These data showed that DK/SH/Y20/06（H4N6） was possibly a re- combinant virus derived from H4N6 subtype, H6N2, H6N5, H3N1 and H1 N2 subtype AIV by complex gene recombination in duck.

Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation

To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.

Molecular evolutionary analysis of gene families encoding DNA recombination and repair proteins and histone demethylases,and their functional implications

Many eukaryotic genes are members of multi-gene families due to gene duplications, which generate new copies that allow functional divergence. However, the relationship between

Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method. Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant, among which 6 samples were from pre-treatment section （influents） and 9 from post-treatment section （effluents）, were analyzed for estrogenic activity by cellar bioassay. Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase, were used to measure the estrogen-like compounds in the farm wastewater treatment plant. Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition. By comparison with a standard curve for 17β-estradiol （E2）, estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent. The estrogenic potency in water samples from the effluents was significantly lower than that in the influents, and 7 water samples had less detectable limit in the total of 9 samples. Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

Recombinant adeno-associated virus delivered human thioredoxin-PR39 prevents hypoxia-induced apoptosis of ECV304 cells

Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.

Osteogenic potential of human calcitonin gene-related peptide alpha gene-modified bone marrow mesenchymal stem cells

Background Most of the basic and clinical studies of osteonecrosis of the femoral head （ONFH） are restricted to bone tissues only, whereas various systems are involved in the onset and development of ONFH, including nervous system. Peptidergic nerve participates in the neuronal regulation of bone metabolism and anabolism, and plays key roles in the growth, repair and reconstruction of bone. Calcitonin gene-related peptide （CGRP）, which is secreted by peptidergic nerve, is the main mediator of bone metabolism. It dramatically promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells （BMSCs）. Additionally, it enhances the osteoblast mass and the rate of osteoblast formation, and reduces the bone resorption by acting on osteoblasts and osteoclasts. Hence, we aimed to construct recombinant retrovirus vector pLNCX2-hCGRPα and to investigate the proliferation and osteogenic potential of hCGRPα-producing BMSCs （BMSCs/pLNCX2-hCGRPα） after virus infection. Methods The constructed recombinant retrovirus vector pLNCX2-hCGRPα was transfected into PT67 packaging cells by lipofectamine 2000. Virus was collected for BMSCs infection. The mRNA and protein expression of hCGRPα was examined by reverse transcription polymerase chain reaction （RT-PCR） and Western blotting, respectively. The cell proliferation was determined by methyl thiazoleterazolium （MTT） assay. The osteogenic potential of BMSCs was evaluated by alkaline phosphatase （ALP） activity. Results Both mRNA and protein expression of hCGRPα was detected in BMSCs/pLNCX2-hCGRPα cells. These cells exhibited significantly elevated proliferation and ALP value as compared with control BMSCs （P 〈0.05）. Conclusion BMSCs/pLNCX2-hCGRPα cells could stably express hCGRPα and showed promoted proliferation ability and osteogenic potential as compared with control BMSCs.

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

OBJECTIVE： Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine（TCM） drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus（r AAV） vectors has not been attempted. METHODS： We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS： The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin（TCS） gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION： Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

Further evidence for paternal DNA transmission in gynogenetic grass carp

Gynogenesis is an important breeding method in aquaculture and has been widely applied to many fish species.If gynogenetic progenies are to inherit paternal partial genomic DNA,this will increase genetic variation and will provide a useful outcome for breeding.In this study,we investigated the genetic variation in homeobox(Hox)gene clusters(Hox A4 a,Hox A9 a,Hox A11 b,Hox B1 b,Hox C4 a,Hox C6 b,and Hox D10 a)among koi carp(Cyprinus carpio haematopterus,KOC;the stimulation sperm source),grass carp(Ctenopharyngodon idellus),and gynogenetic grass carp(GGC).We found paternal DNA(a special DNA fragment and Hox C6 b)derived from KOC and a recombinant gene belonging to Hox C6 b in GGC.We are the first to report the recombinant Hox C6 b in GGC.Our study provides further evidence for paternal DNA transmission to gynogenetic progenies,which is a finding with great significance for the genetic breeding of fish.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Over the last decades,much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology.The breakthrough has been made in recent years with the advent of sequence-specific endonucleases,especially zinc finger nucleases（ZFNs）,transcription activator-like effector nucleases（TALENs） and clustered regularly interspaced short palindromic repeats（CRISPRs） guided nucleases（e.g.,Cas9）.In higher eukaryotic organisms,site-directed mutagenesis usually can be achieved through non-homologous end-joining（NHEJ） repair to the DNA double-strand breaks（DSBs） caused by the exogenously applied nucleases.However,site-specific gene replacement or genuine genome editing through homologous recombination（HR） repair to DSBs remains a challenge.As a proof of concept gene replacement through TALEN-based HR in rice（Oryza sativa）,we successfully produced double point mutations in rice acetolactate synthase gene（OsALS） and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations.After ballistic delivery into rice calli of TALEN construct and donor DNA,nine HR events with different genotypes of OsALS were obtained in T_0 generation at the efficiency of 1.4%—6.3%from three experiments.The HRmediated gene edits were heritable to the progeny of T_1 generation.The edited T_1 plants were as morphologically normal as the control plants while displayed strong herbicide resistance.The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

Inhibition of vascular smooth muscle cell proliferation by in troduction of retinoblastoma gene via a recombinant adenovirus vector

This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.

An Experimental Study on the Flexibility of Prevention against Thrombosis Following Mechanical Valve Replacement by tPA Gene Transduction

Objectives Use a gene suture immersed recombinant tissue-type plasminogen activator （r-tPA）expression plasmid to transduce myocardia to prevent the thrombosis after mechanical tricuspid valve replacement in pigs. Methods A r-tPA gene plasmid was constructed and conjugated to a novel cationic phosphonolipid and a r-tPA gene suture was made. Eighteen pigs were selected and divided into two groups at randomization. There were 9 pigs in the experimental group and 9 in the control group, all the 18 pigs＇ tricuspids were replaced with mechanical valves. The gene threads were sutured into the right ventricular walls near mechanical valves and an ultrasound was used on the surfaces of the right ventricular walls for the gene transfer in the experimental group. Coagulative function, D-dimer level of the blood and the thrombosis on the surfaces of the valves were observed. Results r-tPA gene plasmid was successfully constructed and r-tPA protein was expressed in the ventricular cells around the gene sutures. D-dimer reached its peak level （ 1.67 ±0. 79） μg · mL^-1 in 1 week after operation in two groups, but it decreased to preoperation level thereafter in control group and kept on the high level and reincreased to a new high level （ 1.89 ± 0.79 ） μg · mL^-1 until the end of the experiment in experimental group. The thromboses around the valves were found in all the control group （100%） but only 1 （ 11.11% ） case in experimental group. There were no changes in prothrombin time pre and post operation in two groups. Conclusions Using gene suture immersed r-tPA expression plasmid to transduce myocardia might be a best substitution for life long anti-coagulation therapy for the patients, who underwent operation.

Antitumor effects of human interferon-alpha 2b secreted by recombinant bacillus Calmette-Guérin vaccine on bladder cancer cells

Objective:Our objective was to construct a recombinant bacillus Calmette-Guérin vaccine(rBCG) that secretes human interferon-alpha 2b(IFNα-2b) and to study its immunogenicity and in vitro antitumor activity against human bladder cancer cell lines T24 and T5637.Methods:The signal sequence BCG Ag85B and the gene IFNα-2b were amplified from the genome of BCG and human peripheral blood,respectively,by polymerase chain reaction(PCR).The two genes were cloned in Escherichia coli-BCG shuttle-vector pMV261 to obtain a new recombinant plasmid pMV261-Ag85B-IFNα-2b.BCG was transformed with the recombinant plasmid by electroporation and designated rBCG-IFNα-2b.Mononuclear cells were isolated from human peripheral blood(PBMCs) and stimulated with rBCG-IFNα-2b or wild type BCG for 3 d,and then cultured with human bladder cancer cell lines T24 and T5637.Their cytotoxicities were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay.Results:BCG was successfully transformed with the recombinant plasmid pMV261-Ag85B-IFNα-2b by electroporation and the recombinant BCG(rBCG-IFNα-2b) was capable of synthesizing and secreting cytokine IFNα-2b.PBMC proliferation was enhanced significantly by rBCG-IFNα-2b,and the cytotoxicity of PBMCs stimulated by rBCG-IFNα-2b to T24 and T5627 was significantly stronger in comparison to wild type BCG.Conclusions:A recombinant BCG,secreting human IFNα-2b(rBCG-IFNα-2b),was constructed successfully and was superior to control wild type BCG in inducing immune responses and enhancing cytotoxicity to human bladder cancer cell lines T24 and T5637.This suggests that rBCG-IFNα-2b could be a promising agent for bladder cancer patients in terms of possible reductions in both clinical dosage and side effects of BCG immunotherapy.

Genome Editing:From Drosophila to Non-Model Insects and Beyond

Insect is the largest group of animals on land.Many insect species inflict economical and health losses to humans.Yet many more benefit us by helping to maintain balances in our ecosystem.The benefits that insects offer remain largely untapped,justifying our continuing efforts to develop tools to better understand their biology and to better manage their activities.Here we focus on reviewing the progresses made in the development of genome engineering tools for model insects.Instead of detailed descriptions of the molecular mechanisms underlying each technical advance,we focus our discussion on the logistics for implementing similar tools in non-model insects.Since none of the tools were developed specific for insects,similar approaches can be applied to other non-model organisms.

Labeling embryonic stem cells with enhanced green fluorescent protein on the hypoxanthineguanine phosphoribosyl transferase locus

To label embryonic stem (ES) cells with enhanced green fluorescent protein (EGF P) on the hypoxanthineguanine phosphoribosyl transferase (HPRT) gene locus for t he first time to provide a convenient and efficient way for cell tracking and ma nipulation in the studies of transplantation and stem cell therapy Methods Homologous fragments were obtained by polymerase chain reaction (PCR), from whic h the gene targeting vector pHPRT EGFP was constructed The linearized vector was introduced into ES cells by electroporation The G418 r6TG r cell clones were obtained after selection with G418 and 6TG media The integration patterns of these resistant cell clones were identified with Southern blotting Results EGFP expressing ES cells on the locus of HPRT were successfu lly generated They have normal properties, such as karyotype, viability and di fferentiation ability The green fluorescence of EGFP expressing cells was main tained in propagation of the ES cells for more than 30 passages and in different iated cells Cultured in suspension, the 'green' ES cells aggregated and forme d embryoid bodies, retaining the green fluorescence at varying developmental sta ges The 'green' embryoid bodies could expand and differentiate into various t ypes of cells, exhibiting ubiquitous green fluorescence Conclusions This generation of 'green' targeted ES cells is described in an efficient proto col for obtaining the homologous fragments by PCR Introducing the marker gene in the genome of ES cells, we should be able to manipulate them in vitro and use them as vehicles in cell replacement therapy as well as for other biomedical a nd research purposes