Target Gene Fragment Research Articles

Establishment of a recombinase-aided isothermal amplification technique to detect Schistosoma japonicum specific gene fragments

To establish a novel method for the detection of Schistosoma japonicum specific gene fragments by recombinase aided isothermal amplification (RAA). The gene fragment SjG28 of S. japonicum was selected as the target gene fragment to be detected, and the primers were designed according to the mechanism of RAA reaction. The reaction of isothermal amplification of S. japonicum was established and optimized. Then this method was applied to amplify and detect the specific gene fragment in the gradient diluent SjG28-recombiant plasmids and different concentrations of S. japonicum genomic DNA to estimate the sensitivity of this method. The samples were also detected by polymerase chain reaction (PCR) in parallel as control. This method was applied to detect the genomic DNA of S. mansoni, Ascaris lumbricoides, and Ancylostoma duodenale to evaluate the specificity. The specific gene fragment was amplified from genomic DNA of adult worms and eggs of S. japonicum by recombinase aided isothermal amplification reaction established in this study. The reaction can be completed within 30 minutes and the minimum detectable template was 20 copies of plasmids or 0.5 ng of genomic DNA per microliter. Other parasites'genomic DNAs, such as S. mansoni, A. lumbricoides, An. duodenale and healthy human blood genomic DNA were not able to be detected by this method. A novel method for the detection of S. japonicum specific gene fragments by recombinase aided isothermal amplification is established in this study, which can be carried out conveniently and rapidly with a considerable sensitivity and specificity, showing the prospect for application in the diagnosis of schistosomiasis japonica.

Molecular characterization of Babesia microti seroreactive antigen 5-1-1 and development of rapid detection methods for anti-B. microti antibodies in serum

Babesiosis has become a new global threat impacting human health, and most human babesiosis cases are caused by Babesia microti. Until now few antigens of B. microti have been described which can be used for the diagnosis of human babesiosis. In the present study, we report on the bioinformatic analysis, cloning and expression of the sequence encoding the B. microti seroreactive antigen 5-1-1 to investigate its potential incorporation in serologic diagnostic tools for babesiosis. Bioinformatic analysis and recombinant gene expression were performed to molecularly characterize seroreactive antigen 5-1-1. Enhanced chemiluminescence (ECL)-Western blot methods were used to detect specific antibodies in infected mice. Immunofluorescence antibody assays (IFA) were performed to detect the localization of BmSA5-1-1 in B. microti parasites. ELISA and immunochromatographic (ICT) tests were developed using recombinant BmSA5-1-1 to evaluate its potential use in rapid detection methods for B. microti antibodies and for the diagnosis of babesiosis. A recombinant expression plasmid was constructed by inserting the target gene fragment in the pET28a vector after double digestion with BamHI and XhoI restriction enzymes. The recombinant BmSA5-1-1 protein was expressed in Escherichia coli (rBmSA5-1-1) and purified by means of Ni-nitrilotriacetic acid (NTA) agarose columns. Polyclonal antibodies were generated against rBmSA5-1-1. Based on indirect immunofluorescence assay results, BmSA5-1-1 appeared to localize on the surface of B. microti. ELISA tests using the rBmSA5-1-1 antigen detected specific antibodies from infected mice as early as 4 days post-infection. Our results indicate that the two methods we developed can detect specific antibodies in mice at different stages of infection with sensitivities of 100% (rBmSA5-1-1 ELISA) and 90% (ICT). The specificity of the two methods was 100%. Sera of patients suffering from other closely related parasitic diseases, such as malaria and toxoplasmosis, produced negative results. In conclusion, seroreactive antigen 5-1-1, a member of the BMN1 protein family, is expressed on the outer surface of B. microti and is a promising candidate antigen for the early diagnosis of babesiosis. rBmSA5-1-1 ELISA and ICT methods show good potential for detecting specific antibodies in mice at different stages of infection.

Construction of Overexpression Vector of CONSTANS Gene Plant in <i>Brassica napus</i> and Production of Transgenic Plants

The CONSTANS target gene from Brassica napus was cloned, and the full length amplified primer of it was designed and amplified on the sequence released on NCBI. The target gene fragments were sequenced and the restriction loci were analyzed. The enzyme primers (BamHⅠ, SacⅠ) were designed, and the target gene fragments were connected to the eukaryotic expression vector pBI121 through BamHⅠ and SacⅠ double enzyme digestion. PCR identification result indicated that PBI121+CONSTANS overexpression vector was successfully constructed. The overexpression vector was transformed into the competent EHA105 cell, and the seeds of T0 were infected by Arabidopsis thaliana . The positive plants were screened by MS solid medium containing Cara (50 mg/L). PCR identification showed that PBI121+CONSTANS overexpression vector was successfully introduced into Arabidopsis thaliana and 12 transgenic positive seedlings were obtained, which did the preparation for the gene function analysis.

Construction of prototrophic glutathione-high-producing yeast strain mediated by CRISPR/Cas9

Glutathione (GSH), a non-protein thiol product with various biological activities, has been widely used in pharmaceutical and food industries. Recently, genetic engineering becomes an important strategy for obtaining GSH-high-producing strains. However, auxotrophic selection markers used may result in reduced cell growth or GSH production. In the present study, clustered regularly interspaced short palindromic repeats (CRISPR) and Cas9-associated-system (CRISPR-Cas), in which gRNA expression constructs and homologous DNA fragments of target genes were co-transformed into Saccharomyces cerevisiae cells, was used for the construction of the prototrophic strain derived from the engineered auxotrophic strain W303-1b/FGP. As a result, the prototrophic strain W303-1b/FGPPT showed a significantly shorter culture cycle compared with the auxotrophic strain. Furthermore, chemically defined medium could be used to culture strain W303-1b/FGPPT that might have great interests in industrial fermentation.

Study of gene mutation in 62 hemophilia A children

Objective: To analyze the mutation type of FⅧ gene in children with hemophilia A and to explore the relationship among hemophilia gene mutation spectrum, gene mutation and clinical phenotype. Method: Sixty-two children with hemophilia A from Department of Pediatric Hematology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology between January 2015 and March 2017 were enrolled. All patients were male, aged from 4 months to 7 years and F Ⅷ activity ranged 0.2%-11.0%. Fifty cases had severe, 10 cases had moderate and 2 cases had mild hemophilia A. DNA was isolated from peripheral blood in hemophilia A children and the target gene fragment was amplified by PCR, in combination with the second generation sequencing, 22 and 1 introns were detected. Negative cases were detected by the second generation sequencing and results were compared with those of the international FⅧ gene mutation database. Result: There were 20 cases (32%) of intron 22 inversion, 2 cases (3%) of intron 1 inversion, 18 cases (29%) of missense mutation, 5 cases (8%) of nonsense mutation, 7 cases (11%) of deletion mutation, 1 case(2%)of splice site mutation, 2 cases (3%) of large fragment deletion and 1 case of insertion mutation (2%). No mutation was detected in 2 cases (3%), and 4 cases (7%) failed to amplify. The correlation between phenotype and genotype showed that the most common gene mutation in severe hemophilia A was intron 22 inversion (20 cases), accounting for 40% of severe patients, followed by 11 cases of missense mutation (22%). The most common mutation in moderate hemophilia A was missense mutation (6 cases), accounting for 60% of moderate patients. Conclusion: The most frequent mutation type in hemophilia A was intron 22 inversion, followed by missense mutation, again for missing mutation. The relationship between phenotype and genotype: the most frequent gene mutation in severe hemophilia A is intron 22 inversion, followed by missense mutation; the most frequent gene mutation in medium hemophilia A is missense mutation.

Use of a Virus Gene Silencing Vector for Maize Functional Genomics Research.

Virus-induced gene silencing (VIGS) is a genetic technology that exploits the RNA-mediated defense against virus. The method has great potential for plant reverse genetics as it could knock down gene expression in a rapid way, which is triggered by a replicating viral genome engineered to carry a fragment of host gene to be silenced. A number of efficient VIGS vectors are available for dicots, such as for model plant Nicotiana benthamiana; however, only a few of VIGS vectors for monocotyledonous cereal crops. Here, we describe the method for the use of a newly developed VIGS vector based on a maize-infecting Cucumber mosaic virus (CMV) strain ZMBJ-CMV for maize. The RNA2 of ZMBJ-CMV was modified as a vector pCMV201-2bN81 having multiple cloning sites for the insert of 100-300bp fragment of target gene. Using a method of vascular puncture inoculation of maize seeds with crude sap prepared from Agrobacterium-infiltrated N. benthamiana leaves, silencing of target genes could be obtained in 4weeks.

Effect of mmu-miR-30b on mouse FoxO3 mRNA expression

Objective To analyze the direct interaction between mmu-miR-30b and mouse FoxO3 (mFoxO3) mRNA. Methods Three target gene fragments, which were respectively 402, 123 and 299 bp in length, were amplified from mouse cDNA by PCR using specific primers and site-direct mutant primers. A complete mutant fragment was obtained by joining together the 123 and 299 bp fragments. Recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed through inserting wild and mutant fragments of mFoxO3 into pmirGLO vector, respectively. HEK 293T cells were respectively co-transfected with the constructed recombinant plasmids and mmu-miR-30b/mmu-miR-30b inhibitor. Dual-luciferase reporter assay system was used to determine the Firefly-Renilla luciferase activity in different groups. Western blot assay was performed to evaluate the regulatory effect of mmu-miR-30b on mFoxO3 expression. Results Restriction enzyme analysis and gene sequencing showed that the two recombinant plasmids, pmirGLO-mFoxO3 and pmirGLO-mFoxO3 mt, were constructed successfully. The activity of Firefly-Renilla luciferase in HEK 293T cells transfected with pmirGLO, pmirGLO+ mmu-miR-30b, pmirGLO+ mmu-miR-30b inhibitor, pmirGLO-mFoxO3+ mmu-miR-30b, pmirGLO-mFoxO3+ mmu-miR-30b+ mmu-miR-30b inhibitor, or pmirGLO-mFoxO3 mt+ mmu-miR-30b was 13.18±0.97, 14.35±0.99, 12.46±1.20, 9.55±1.11, 13.71±0.89 and 10.99±0.92, respectively. Compared with pmirGLO+ mmu-miR-30b group, the luciferase activity was significantly decreased in pmirGLO-mFoxO3+ mmu-miR-30b group (P 0.05). In addition, the suppressed Firefly-Renilla luciferase activity in pmirGLO-mFoxO3+ mmu-miR-30b group was restored by mmu-miR-30b inhibitor treatment (P<0.05). Enhancing the expression of mmu-miR-30b could markedly inhibit the expression of mFoxO3 at protien level (P<0.05), and that could be significantly attenuated by mmu-miR-30b inhibitor treatmeat (P<0.05). Conclusion mFoxO3 mRNA is a novel target gene of mmu-miR-30b. There is a direct interaction between mmu-miR-30b and mFoxO3 mRNA. Key words: mmu-miR-30b; Mouse FoxO3; Target gene

Genome Editing in Cotton with the CRISPR/Cas9 System.

Genome editing is an important tool for gene functional studies as well as crop improvement. The recent development of the CRISPR/Cas9 system using single guide RNA molecules (sgRNAs) to direct precise double strand breaks in the genome has the potential to revolutionize agriculture. Unfortunately, not all sgRNAs are equally efficient and it is difficult to predict their efficiency by bioinformatics. In crops such as cotton (Gossypium hirsutum L.), with labor-intensive and lengthy transformation procedures, it is essential to minimize the risk of using an ineffective sgRNA that could result in the production of transgenic plants without the desired CRISPR-induced mutations. In this study, we have developed a fast and efficient method to validate the functionality of sgRNAs in cotton using a transient expression system. We have used this method to validate target sites for three different genes GhPDS, GhCLA1, and GhEF1 and analyzed the nature of the CRISPR/Cas9-induced mutations. In our experiments, the most frequent type of mutations observed in cotton cotyledons were deletions (∼64%). We prove that the CRISPR/Cas9 system can effectively produce mutations in homeologous cotton genes, an important requisite in this allotetraploid crop. We also show that multiple gene targeting can be achieved in cotton with the simultaneous expression of several sgRNAs and have generated mutations in GhPDS and GhEF1 at two target sites. Additionally, we have used the CRISPR/Cas9 system to produce targeted gene fragment deletions in the GhPDS locus. Finally, we obtained transgenic cotton plants containing CRISPR/Cas9-induced gene editing mutations in the GhCLA1 gene. The mutation efficiency was very high, with 80.6% of the transgenic lines containing mutations in the GhCLA1 target site resulting in an intense albino phenotype due to interference with chloroplast biogenesis.

A simple and effective method for assessing chromatin diminution values in copepods using qPCR

The value of chromatin diminution (CD) in different species of freshwater cyclopoid copepods can differ significantly. The biological and evolutionary roles of these differences remain unclear. To expand the knowledge on CD distribution and magnitude in this group of copepods, a quick method for its evaluation was required. This study proposes a simple approach for CD assessment in copepods using quantitative realtime PCR (qPCR). The magnitude of changes in the genome size was assessed by comparing fluorescence curves of qPCR fragments of target genes for pre- and post-diminution materials. The method was tested on four cyclopoid copepods species. In Cyclops kolensis, CD was assessed as 95.3 ± 1.2; in Acanthocyclops vernalis it was assessed at 94.6 ± 0.8%; at C. insignis, it was 82.3 ± 5.2%; and for the first time, CD was found in Megacyclops viridis at 91.1 ± 2.6%. The advantages of our approach are its rapidity, simplicity and minimal requirements of materials studied.

Early detection of Trichinella spiralis DNA in the feces of experimentally infected mice by using PCR

The aim of this study was to detect Trichinella spiralis DNA in mouse feces during the early stages of infection using PCR. The target gene fragment, a 1.6kb repetitive sequence of T. spiralis genome, was amplified by PCR from feces of mice infected with 100 or 300 larvae at 3–24h post infection (hpi) and 2–28dpi. The sensitivity of PCR was 0.016 larvae in feces. The primers used were highly specific for T. spiralis. No cross-reactivity was observed with the DNA of other intestinal helminths. T. spiralis DNA was detected in 100% (12/12) of feces of mice infected with 100 or 300 larvae as early as 3hpi, with the peak detection lasting to 12–24hpi, and then fluctuating before declining gradually. By 28dpi, the detection rate of T. spiralis DNA in feces of the two groups of infected mice decreased to 8.33% and 25%, respectively. PCR detection of T. spiralis DNA in feces is simple and specific; it might be useful for the early diagnosis of Trichinella infection.

Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9.

Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.

Rapid identification and quantitation for oral bacteria based on short-end capillary electrophoresis

High-speed capillary electrophoresis (HSCE) is a promising technology applied in ultra-rapid and high-performance analysis of biomolecules (such as nucleic acids, protein). In present study, the short-end capillary electrophoresis coupled with one novel space domain internal standard method (SDIS) was employed for the rapid and simultaneous analysis of specific genes from three oral bacteria (Porphyromonas gingivalis (P.g), Treponema denticola (T.d) and Tannerela forsythia (T.f)). The reliability, reproducibility and accuracy properties of above mentioned SDIS method were investigated in detail. The results showed the target gene fragments of P.g, T.d and T.f could be precisely, fast identified and quantitated within 95s via present short-end CE system. The analyte concentration and the ratio of space domain signals (between target sample and internal standard sample) featured a well linear relationship calculated via SDIS method. And the correlation coefficients R2 and detection limits for P.g, T.d, T.f genes were 0.9855, 0.9896, 0.9969 and 0.077, 0.114 and 0.098ng/μl, respectively.

Simultaneous detection of double gene-specific methylation loci based on hairpin probes tagged with electrochemical quantum dots barcodes

A novel label-free and PCR-free electrochemical method is demonstrated for simultaneous evaluation of double gene-specific methylation loci in a single-tube experiment without numerous hybridization and heating processes. The method composes three units: two electrochemical quantum dots tagged dual-functional hairpin probes (HP1-CdS and HP2-PbS) for methylation loci recognition, and capture probes modified magnetic beads (MB-CP) as isolation vehicle. A p53 gene fragment with two methylation loci was selected as a target model. After pretreatment with sodium bisulfite, samples were simply incubated with HP1-PbS, HP2-CdS, and MB-CP conjugates. The resultant MBs were then isolated and stripping analyzed by square wave voltammetry (SWV). From the SWV responses of the QDs barcodes hybridized on MBs, the methylation pattern and level of target gene fragment were clearly identified. The linear range was 5.0–125.0nM with a limit of detection (LOD) of 1.3nM for locus A which was tagged by PbS, and 0.5–125.0nM with a LOD of 0.38nM for locus B which was tagged by CdS. The method showed satisfactory accuracy and testing recovery. In comparison with the current methods, the proposed method is convenient and can evaluate two gene-specific methylation in a single-tube experiment within 2.5h. With regard to the advantages of facility, low-cost, and ease of operation, the method can be a potential platform for identification of DNA methylation status in a simple way.

Influence of the PCR artifacts on the genotyping efficiency by the microsatellite loci using native polyacrylamide gel electrophoresis

The formation of different types of the artifacts in the amplification reaction with using different classes of the molecular-genetic markers (Indel and SSR) was studied. It was shown that DNA heteroduplexes formed during amplification of heterozygous samples, as fragments of target genes and microsatellite loci. During the electrophoresis in native polyacrylamide gel of the amplification products of homozygous samples for microsatellite loci it was revealed specific additional fragments that do not belong to the class of heteroduplex DNA. It was supposed, that the additional fragments belong to a special type of homoduplex DNA – non-linear homoduplexes. The analysis revealed that the formation of a non-linear homoduplex DNA takes place on the 20–25 cycle of the PCR, both at the amplification of the individual samples, and individual DNA fragments that was cut out from the gel.

Investigation of a miRNA-Induced Gene Silencing Technique in Petunia Reveals Alterations in miR173 Precursor Processing and the Accumulation of Secondary siRNAs from Endogenous Genes.

MIGS (miRNA-induced gene silencing) is a straightforward and efficient gene silencing technique in Arabidopsis. It works by exploiting miR173 to trigger the production of phasiRNAs (phased small interfering RNAs). MIGS can be used in plant species other than Arabidopsis by co-expression of miR173 and target gene fragments fused to an upstream miR173 target site. However, the efficiency and technical mechanisms have not been thoroughly investigated in other plants. In this work, two vectors, pMIGS-chs and pMIGS-pds, were constructed and transformed into petunia plants. The transgenic plants showed CHS (chalcone synthase) and PDS (phytoene desaturase) gene-silencing phenotypes respectively, indicating that MIGS functions in petunia. MIGS-chs plants were used to investigate the mechanisms of this technique in petunia. Results of 5′- RACE showed that the miR173 target site was cleaved at the expected position and that endogenous CHS genes were cut at multiple positions. Small RNA deep sequencing analysis showed that the processing of Arabidopsis miR173 precursors in MIGS-chs transgenic petunia plants did not occur in exactly the same way as in Arabidopsis, suggesting differences in the machinery of miRNA processing between plant species. Small RNAs in-phase with the miR173 cleavage register were produced immediately downstream from the cleavage site and out-of-phase small RNAs were accumulated at relatively high levels from processing cycle 5 onwards. Secondary siRNAs were generated from multiple sites of endogenous CHS-A and CHS-J genes, indicating that miR173 cleavage induced siRNAs have the same ability to initiate siRNA transitivity as the siRNAs functioning in co-suppression and hpRNA silencing. On account of the simplicity of vector construction and the transitive amplification of signals from endogenous transcripts, MIGS is a good alternative gene silencing method for plants, especially for silencing a cluster of homologous genes with redundant functions.

Expression, purification and identification of Pla a1 in a codon-optimized Platanus pollen allergen.

The present study aimed to express, purify and identify the major allergen gene, Pla a1, in Platanus pollen. According to previous studies, the major gene sequences of the Pla a1 allergen were obtained and codon optimization and synthesis of the genome were performed using DNAStar software. Following binding of the target gene fragment and the pET-44a vector, the JM109 cells were transfected to produce positive clones. The vectors were then transformed into Escherichia coli Rosetta cells to induce the expression of the target protein. The exogenous protein was purified using affinity chromatography and was identified by western blot analysis. Pla a1, the major allergen protein in Platanus pollen, was successfully isolated and this exogenous protein was purified using affinity chromatography. The present study was the first, to the best of our knowledge, to obtain expression of the allergen recombinant protein, Pla a1, fused with a Strep-TagII via codon optimization and provided the basis for the preparation of allergens with high purity, recombinant hypoallergenic allergens and allergen nucleic acid vaccines.

Construction of a mutant strain of Streptococcus mutans with clpC-deletion to study the role of clpC ;gene in genetic competence

Objective To construct a mutant strain of Streptococcus mutans (S.mutans) with clpC-deletion and to investigate the role of clpC gene in genetic competence. Methods The fragment of clpC gene and the kanamycin resistant cassette flanked by two loxP sites were amplified by PCR. The purified fragment of clpC gene was cloned into pMD-19T simple vector to construct pCKX1. The pCKX1 vector was digested with ClaⅠ/EcoRⅠ, then blunted and introduced into lox71-KMR-lox66 to obtain pCKX2 vector via homologous recombination. The pCKX2 vector was linearized with SalⅠ and transformed into S. mutans UA159 strain. The positive strains constructed via homologous recombination were screened with kanamycin and transformed with the thermosensitive plasmid pCrePA. The KMR cassette was excised after incubating at 30℃ for 48 hours. Then the pCrePA plasmid was removed after overnight incubating at 37℃ for the preparation of clpC-deletion mutant. Total RNA were extracted from the S. mutans UA159 strain and the clpC-deletion mutant strain respectively, and then reverse transcribed into first strand cDNA. The target gene fragments were amplified by RT-PCR and analyzed by the agarose gel electrophoresis and sequencing. After being verified by PCR and sequencing, the S. mutans UA159 strain and the clpC-deletion mutant strain were respectively transformed with E. coli-S.mutans shuttle vector pDL276 to observe the competence development induced by the competence-stimulating peptide (CSP). Results The PCR and sequencing results showed that the pCKX2 vector and the mutant strain with clpC-deletion were constructed successfully via homologous recombination. No clpC gene was detected in the clpC-deletion mutant as indicated by RT-PCR analysis. The formation of competent clpC-deletion mutant was delayed and the competence state was prolonged as compared with its parent strains. Conclusion The clpC gene negatively regulated the formation of competent S. mutans. Key words: Streptococcus mutans; Competence; clpC; Cre/loxP

Construction and identification of recombinant adenovirus carrying human TIMP-1shRNA gene.

The aim of this study was to construct the recombinant adenovirus carrying human TIMP-1shRNA gene expression system for preliminary identification to lay the foundation for the further study of gene therapy. Using the Adeno-X system, the recombinant adenovirus plasmid pAdeno-X green fluorescent protein (GFP)-tissue inhibitor of metalloprotease (TIMP)-1 small hairpin (1shRNA) was constructed by including the target gene fragment of the TIMP-1shRNA shuttle plasmid pShuttle2-GFP-TIMP-1shRNA and the backbone plasmid pAdeno-X by homologous recombination in Escherichia coli. Recombinant plasmids were transfected into HEK293A cells to package the recombinant adenovirus rvAdeno-XGFP-TIMP-1shRNA. The recombinant adenovirus was identified by polymerase chain reaction, and the viral titer and infection efficiency were detected using GFP. Polymerase chain reaction and restriction endonuclease digestion demonstrated that rvAdeno-XGFP-TIMP-1shRNA had been successfully constructed, which has a strong ability to infect the kidney. The TIMP-1shRNA adenovirus expression vector was successfully constructed using homologous recombination methods.

Advances of lentiviral vectors

慢病毒载体（lentivirus vector, LV）是目前分子及细胞生物实验中非常有效的工具，在基因转染方面有着许多独特的优势，例如，对细胞是否处于有丝分裂期没有特别的要求，基因转染效率高，可容纳较大基因片段等。本文将就慢病毒载体的来源、分子特征及研究进展等进行综述。

Construction of the Lentiviral and Electric Transfection Vectors Encoding Anti-Human CD30 or CD33 Chimeric Antigen Receptor (CAR) Gene and Respective Expression Injurkat Leukemia Cell Line

Adoptive cellular therapy (ACT), as a kind of biological treatment, has played an important role in immunotherapy for malignancies, by which an individual passively acquires the specific immunological rejection to tumor molecular target through cellular infusion, resulting in the suppression or destruction of malignancies. In the early clinical studies, immunotherapies based on tumor-infiltrating lymphocytes (TILs) have demonstrated definitively therapeutic effect on a variety of tumors, especially on melanoma, which underlined feasibility and necessity of ACT in anti-tumor therapies. However, the difficulties on cellular material acquisition, relatively complex cellular constitute, undefined anti-tumor target and unstable efficacy, all of which affected and limited the clinical practice with TILs, in addition to the immune tolerance-inducing behaviors of the tumor itself, such as the immune escape, suppression of the immune effects via peri-tumor microenvironment, down-regulation of MHC to minimize antigen exposure, et al.Chimeric antigen receptor (CAR) has effectively integrated the specific recognition of Ab-Ag and signal transfer function of TCR to form a transmembrane molecule with different functional domains. The expression fragment of CAR could be formed by aligning single chain antibody fragment (scFv) cDNA and costimulatory domain in tandem, which is subsequently transferred via lentiviral vectors to modify autologous T lymphocytes. After gene expression and translation, the CAR has an MHC-independent specific recognition to tumor-associated antigen (TAA), resulting in T cell activation, anti-tumor cytotoxic effort and memory cell differentiation. CAR-modified lymphocytes (CART) are effective ACT tools cells with anti-tumor specificity through gene transfection. Currently, CD19-targeted CART therapy has yielded promising therapeutic effect.Our study selected a lineage-specific CD33 and another tumor-specific CD30 as CAR-associated targets, with which we designed and constructed a series of third generation CAR containing CD28 activation domain and 4-1BB costimulatory domain. The CAR-containing expression vectors were transfected into the Jurkat cell line via lentiviral system and nucleofector electroporation system, respectively.The target gene fragments, anti-CD30-scFv, anti-CD33-scFv, and CD28-4-1BB-CD3ζ sequence encoding T cell activation plus costimulatory signals were amplified from pUC57-CD30-scFv, pUC57-CD33-scFv and pUC57-CD2841BBZ via PCR. The separated scFv and signal sequence were linked into a continuous fragment, CAR-CD30 and CAR-CD33 cDNA, by overlapping PCR, which were subsequently subcloned into a lentiviral expression vector, pCDH-CMV-MCS-EF1-Puro, to construct recombinant lentiviral vector pCDH-CAR30 and pCDH-CAR33; the electric transfection vector pmax-CAR30 and pmax-CAR33 were constructed as well.With virus packaging, the corresponding CARs/GFP/Blank-containing virus were added into Jurkat cell medium, which were immediately centrifuged to facilitate infection; puromycin was used to screen Jurkat strains highly expressing CAR, CAR-modified Jurkat strains were thus harvested one week from antibiotic screening, named as: Jurkat-pCDH-Blank, Jurkat-pCDH-GFP, Jurkat-pCDH-CAR30 and Jurkat-pCDH-CAR33, whose protein samples were then extracted for CAR detection.The Jurkat cells could also be effectively transfected by applying an electrical pulse to momentarily create small pores in cell membranes, with the aid of pmax-CAR30 or pmax-CAR33, moreover, different discharge mode was designed to yield higher cell viability, and otherwise higher transfection efficiency. The fluorescent signal could be observed 24h from electric pulse and protein samples could be extracted at the same time.After one-week screening with puromycin, fluorescence from Jurkat cells infected with GFP-containing virus were detected. Electroporation was used as an alternative procedure to produce CAR-modified Jurkat cell line, and the transfection efficiency was higher than lentiviral system did, approximately 90%, by GFP observation. The expression of CAR-CD30 and CAR-CD33 were also successfully detected by western blot in Jurkat cells, indicating a preliminary and technical basis ahead of in-depth study on ACT and CART agent preparation. DisclosuresNo relevant conflicts of interest to declare.