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Gene editing technology is a genetic operation technology that can modify the DNA sequence at the genomic level. The precision gene editing technology based on CRISPR/Cas9 system is a gene editing technology that is easy to operate and widely used. Unlike the traditional CRISPR/Cas9 system, the precision gene editing technology can perform site-directed mutation of genes without DNA template. This review summarizes the recent development of precision gene editing technology based on CRISPR/Cas9, and prospects the challenges and opportunities of this technology.
Subject(s)
Gene Editing , CRISPR-Cas Systems/genetics , Mutation , GenomeABSTRACT
In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.
Subject(s)
Animals , Zebrafish/genetics , Animals, Genetically Modified/genetics , Green Fluorescent Proteins/metabolism , Zebrafish Proteins/genetics , Promoter Regions, GeneticABSTRACT
PiggyBac is a transposable DNA element originally discovered in the cabbage looper moth (Trichoplusia ni). The T. ni piggyBac transposon can introduce exogenous fragments into a genome, constructing a transgenic organism. Nevertheless, the comprehensive analysis of endogenous piggyBac-like elements (PLEs) is important before using piggyBac, because they may influence the genetic stability of transgenic lines. Herein, we conducted a genome-wide analysis of PLEs in the brown planthopper (BPH) Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), and identified a total of 28 PLE sequences. All N. lugens piggyBac-like elements (NlPLEs) were present as multiple copies in the genome of BPH. Among the identified NlPLEs, NlPLE25 had the highest copy number and it was distributed on five chromosomes. The full length of NlPLE25 consisted of terminal inverted repeats and sub-terminal inverted repeats at both terminals, as well as a single open reading frame transposase encoding 546 amino acids. Furthermore, NlPLE25 transposase caused precise excision and transposition in cultured insect cells and also restored the original TTAA target sequence after excision. A cross-recognition between the NlPLE25 transposon and the piggyBac transposon was also revealed in this study. These findings provide useful information for the construction of transgenic insect lines.
Subject(s)
Animals , Amino Acid Sequence , Animals, Genetically Modified , DNA Transposable Elements/genetics , Hemiptera/genetics , Transposases/geneticsABSTRACT
Essential fatty acids are those that could not be synthesized by the body itself but crucial for health and life. Studies have shown that ω-3 fatty acids may facilitate human physiological functions. Mammals lack ω-3 desaturase gene, and the Δ15 fatty acid desaturase (Δ15 Des) from Caenorhabditis elegans can transform the ω-6 polyunsaturated fatty acids (PUFAs) into ω-3 PUFAs. Transgenic mice expressing Δ15 Des enzyme activity was constructed by using a PiggyBac transposon (PB). Homozygous transgenic mice with stable inheritance was bred in a short time, with a positive rate of 35.1% achieved. The mice were fed with 6% ω-6 PUFAs and the changes of fatty acids in mice were detected by gas chromatography (GC). The expression level of Δ15 Des in mice was detected by quantitative PCR (qPCR) and Western blotting (WB). qPCR and GC analysis revealed that the percentage of positive mice harboring the active gene was 61.53%. Compared with traditional methods, the transformation efficiency and activity of Δ15 Des were significantly improved, and homozygotes showed higher activity than that of heterozygotes. This further verified the efficient transduction efficiency of the PiggyBac transposon system.
Subject(s)
Animals , Mice , Caenorhabditis elegans/genetics , Fatty Acid Desaturases/genetics , Fatty Acids , Fatty Acids, Omega-3 , Mice, TransgenicABSTRACT
Objective:To construct a transposon mutation library and screen new virulence genes of hypervirulent Klebsiella pneumoniae(hvKp). Methods:The transposon mutation library was constructed and treated with human serum. The changes in the abundance of the genes of library mutant strains were analyzed by Transposon sequencing (Tn-seq). Besides, KEGG (kyoto encyclopedia of genes and genomes) annotation and enrichment analysis were performed on the screened genes.Results:A total of 405 genes were screened out according to the abundance of the genes in the library treated with human serum was 20% lower than that without treated, and 351 genes, 86.7% of these genes were conserved in HS11286, NJST258_1, NTUH-K2044 and RJF293. Ten genes existed in strains NTUH-K2044 and RJF293 with high virulence, while these genes were absent in HS11286 and NJST258_1 with low virulence. The mutants with genes such as glycosyl transferase gene wzy, aggregator protein gene wzi and capsule transporter gene wza, which belong to the capsule polysaccharide gene clusters, could not be detected after serum treatment. The abundance of iron carriers gene clusters such as aerobacterin and salmonellin in each library changed less than one time. KEGG annotation results showed that most annotated genes were involved in amino acid metabolism, cofactor and vitamin metabolism, carbohydrate metabolism, etc. Conclusions:Tn-seq is a reliable method to screen functional genes. In this study, 405 candidate virulence genes of hvKp were successfully screened out, providing an experimental basis for further research on the function and regulation mechanism of new virulence genes of hvKp.
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【Objective】 To explore the establishment methods of transgenic human umbilical cord mesenchymal stem cells (hUC-MSCs) overexpressing tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL) based on the transposons, and attempt to apply it on the nude mice mode with glioma. 【Methods】 PiggyBac transposon system specially designed by us was used to prepare non-targeting and Her2-targeting hUC-MSCs that can stably express TRAIL through puromycin screening. The glioma cells expressing firefly luciferase (U87MG-LUC) were injected into the skull of the immunodeficient mice (BALB/c-nu/nu) with 1×106 cells per mouse. After 7 days of injection, the mice transplanted with U87MG were detected with a small animal living imager to determine the size and location of the tumors in skull. Then we injected the glioma-transplantation nude mouse with two kinds of transgenic hUC-MSCs expressing TRAIL (named as untarget-TRAIL and target-TRAIL, respectively), or the non-transgenic hUC-MSCs (all 1×106 cells per mouse) or PBS (named as WT-MSCs and PBS for negative control) respectively, and then monitored the changes of tumor signals by a small animal living imager every week for 3~4 weeks. 【Results】 After six passages to expand the cells, the both transgenic cell lines can stably express TRAIL gene. Their ratio of green fluorescent protein (GFP) positive cells can reach 93%-97%, and the positive ratio of their MSC-specific surface markers still maintained normal (CD34+, CD45+, and HLA-DR+ all <0.1%, CD90>99%, CD73>88%, and CD105 >60%). The median survival time (d) of U87MG-transplanted nude mice in the groups of untarget-TRAIL, target-TRAIL, WT-MSCs, and PBS was 41 vs 39 vs 24 vs 23(P<0.05). 【Conclusion】 The transgenic hUC-MSCs overexpressing TRAIL gene can significantly prolong the survival time of nude mice with brain glioma.
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Objetive: To investigate the long-term expression of the piggyback (PB) transposon system expressing human interleukin-6 (IL-6), nterleukin-3 (IL-3), interleukin-15 (IL-15), stem cell factor (SCF) and granulocyte-macrophage ctimulang Factor (GM-CSF) genes in the immunodeficient mice, and to provide a simple, long-term and new method for improving the reconstruction of human immune cells in the humanized mouse models. Methods: The PB transposon plasmid (PB-GFP) containing the GFP gene was constructed, and the PB transposon plasmid (PB-5F) contaning human IL-6, IL-3, IL-15, SCF, and GM-CSF genes was constructed. The 293T cells were divided into negative control group (non-transfection), postive control group (transfected with pLVTHM), transient transfection group transfected with PB-GFP plasmid and stable transfection group transfected with PB-GFP plasmid together with transposase plasmid (super-PB)]. The proportions of GFP cells in varions groups were measured by flow cytometry every three days after transfection. The NOD. Cg-Prkdcscid IL2rgun1wj1/SzJ (NCG) mice were divided into transient transfection group and stable transfection group. The mice in transient transfection group were transfected with PB-GFP alone, and the mice in stable transfection group were transfected with PB-5F and super-PB. On the 1st, 4th, 5th and 9th days and the followng every week after the transfection, the blood samples were collected, and the serum was separed; the levels of IL-6, IL-3, IL-15, SCF, and GM-CSF in serum of the mice in various groups were detected by ELISA Results: At 30 d after transfection of PB-GFP, the percentage of GFP1 cells of the mice in stable transfection group (4. 61% + 0.42%) was significantly higher than those in postive control group (0.58% +0.05%) and transient transfection group (0.86%+ 0.10%) (P<0.05). At 30 d after transfection of PB-5F plasmid, the levels of serum IL-6, IL-15 and GM-CSF of the NCG mice in stable transfection group were significantly higher than those in transient transfection group (P
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@#Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.
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Ralstonia solanacearum strain PRS-84 used in this study was isolated from diseased Pogostemon cablin plants in our previous study.The competent cells of R.solanacearum strain PRS-84 were transformed by electroporation with Tn5 transposon and then were plated on TTC agar plates containing kanamycin to select for kanamycin-resistant colonies.The detection of kanamycin-resistant gene in kanamycin-resistant colonies was performed by PCR.Further,the flanking fragments of Tn5 transposon insertion site in the mutants were amplified by inverse PCR,and the flanking fragments were sequenced and analyzed.The results indicated that the kanamycin-resistant colonies were obtained in the transformation experiment of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon.A specific band of approximately 700 bp was amplified by PCR from kanamycin-resistant colonies.The flanking sequences of Tn5 transposon insertion site in the transformants were obtained by inverse PCR.After sequencing and sequence analysis of Tn5 transposon insertion site in mutants,we preliminarily speculated that the Tn5 transposon inserted in the typ A gene,rec O gene and gid A gene in three mutants,respectively.A random mutagenesis system of R.solanacearum strain PRS-84 by electroporation with Tn5 transposon has been established,and the Tn5 insertion mutants have been obtained.This study might facilitate the creation of mutant library and the discovery of the virulence gene of R.solanacearum isolated from P.cablin.
Subject(s)
DNA Transposable Elements , Electroporation , Genes, Bacterial , Mutagenesis, Insertional , Pogostemon , Microbiology , Ralstonia solanacearum , Genetics , VirulenceABSTRACT
With the completion of large-scale genome sequencing of human beings and other organisms, understanding the expression of control elements on the genome has become an important research task in the post-genome era. The enhancer trapping technology is an effective method for identifying enhancer elements in the genome and understanding its mechanism for gene expression regulation. In this study, we selected the stable enhancer trapping line TK4 (head and trunk specific GFP expression), which is generated with the mediation of Tol2 transposon system, and analyzed the trapped enhancers with the techniques of Splinkerette PCR (sp-PCR), in situ hybridization and comparative genomics. We crossed F1 individuals of TK4 line with wild-type zebrafish, collected fertilized eggs, and then detected the expression pattern of green fluorescent protein reporter gene by fluorescence microscopy at six different developmental stages, 6 hpf (hour post fertilization), 24 hpf, 48 hpf, 3 dpf (day post fertilization), 4 dpf and 5 dpf . The zebrafish genome flank sequence near the insertion site of Tol2 transposon was cloned by sp-PCR, and the results revealed that the insertion located at the position 27749253 of chromosome 23, and the transgene inserted reversely inside the intron 1 of rps26 gene. Within the 100 kb region of the insertion site, totally, seven genes including arf3a, wnt10b, wnt1, rps26, IKZF4, dnajc22 and lmbr1l were identified. Comparative genomic analysis by VISTA program revealed that there were two potential enhancer elements in the downstream of rps26 gene, which were conserved non-coding sequence (CNS) 1 and CNS2. The results of in situ hybridization showed that two transcripts of rps26 gene were maternal expression, the expression of rps26-201 in zygote was earlier than that of rps26-001, and the GFP signal of TK4 line zebrafish was not detectable before 6hpf, the expression patterns of rps26 and GFP at the late stages display similarity, and also represent differences, which suggested that the expression of rps26 and GFP may be controlled by the same enhancer, and also by the different enhancer, and two potential enhancers (CNS1 and CNS2) may play a differential regulation roles on the spatial and temporal expression of nearby genes (including rps26). In this study, we successfully obtained two potential enhancers near rps26 gene for the first time, which laid a foundation for further study of the regulation mechanism between these two enhancers and nearby genes in the genome, and the combination technique used in this study also provides a reference for enhancer analysis.
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Spt proteins are defined as a large family of transcription regulators of the yeast Saccharomyces cerevisiae. They are crucial components of the SAGA complex that regulates transcription through interaction with the TATA box in the upstream region of the target genes. About 10% of total gene transcriptions are related to Spt proteins and these genes are highly related to environmental stress response. Such vast regulation network and complex mechanisms have become a hotspot. Spt proteins are also important to suppress transposon-induced mutations, being a switch on regulation of transposon behaviors and adaptive evolution of Saccharomyces cerevisiae. Besides that, some Spt proteins are directly involved in regulating unsaturated lipid acids synthesis, which could remold the cell membrane to resist environmental stresses. Here, we review Spt proteins, the advances in Spt proteins study, and their potential applications in improving yeast's stress resistance through transcription regulation, transposon activity regulation and cell membrane alternation.
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Objective:To investigate the effect of co-expression of sleeping beauty(SB)transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes(NOD)mice, and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice.Methods:The splenocytes were extracted from spleen of the C57BL6 mice and cultured.The splenocytes were divided into control group,empty vector group and therapy group.The cells in control group didn't receive any treatment,the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase,and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase.After 48 h of infection, the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR. The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group,empty vector group and therapy group;there were six mice in each group;once a week and six times.The mice were killed 4 weeks after injection,the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA,and the proportions of CD4+IL-10+,CD4+IFN-γ+,Th17 and Treg cells in the splenocytes were detected by flow cytometry.Results:Compared with control group and empty vector group,the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased (F=72.71,P<0.05);the expression level of IL-10 in serum of the NOD mice was increased(F=8.89,P<0.05);the proportions of CD4+IL-10+ cells and Treg cells were significantly increased(F=72.09,P<0.05;F=12.98,P<0.05);the proportion of Th17 cells was decreased(F=6.39,P<0.05).The proportion of CD4+IFN-γ+ cells had no significant differences between various groups(F=2.72,P>0.05).Conclusion:The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice,increase the proportion of CD4+IL-10+ cells,decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes,which can regulate the balance of Th1/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.
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To investigate the effect of co-expression of sleeping beauty (SB) transposon and IL-10 mediated by adenovirus on the balance of Th17/Treg cells in the splenocytes of the non-obese diabetes (NOD) mice, and to illuminate the possible mechanism of IL-10 in the treatment of NOD mice. Methods: The splenocytes were extracted from spleen of the C57BL6 mice and cultured. The splenocytes were divided into control group, empty vector group and therapy group. The cells in control group didn't receive any treatment, the cells in empty vector group were co-infected with the adenovirus vector without IL-10 gene containing transposon sequence and the adenovirus vector without transposase, and the cells in therapy cells were co-infected with the adenovirus vector containing IL-10 gene and transposon sequence and the adenovirus vector with transposase. After 48 h of infection, the expression leves of IL-10 mRNA in the splenocytes of the mice in various groups were assessed by RT-PCR. The cells of above three groups were subcutaneously injected into popliteal space in right hind leg of the NOD mice in control group, empty vector group and therapy group; there were six mice in each group; once a week and six times. The mice were killed 4 weeks after injection, the expression levels of IL-10 in serum of the NOD mice in various groups were assessed by ELISA, and the proportions of CD4 +IL-10+, CD4 + IFN-γ+, Th17 and Treg cells in the splenocytes were detected by flow cytometry. Results: Compared with control group and empty vector group, the expression level of IL-10 mRNA in splenocytes of the C57BL6 mice in therapy group was increased (F=72.71, P0.05). Conclusion: The co-expression of SB transposon and IL-10 can significantly increase the IL-10 level in serum of the NOD mice, increase the proportion of CD4 + IL-10+ cells, decrease the proportion of Th17 cells and increase the proportion of Treg cells in the splenocytes, which can regulate the balance of Thl/Th2 and Th17/Treg cells and play a therapeutic effect in the NOD mice.
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Objective@#To investigate the distribution of blaKPC gene in Lishui and to analyze the molecular epidemiological characteristics of Klebsiella pneumoniae (K.pneumoniae) blaKPC gene.@*Methods@#From 2010 to 2016, all of the non-repetitive K. pneumoniae carbapenemase (KPC)-producing isolates in Lishui Municipal Central Hospital were collected. They were identified with VITEK 2 Compact system and typed by multilocus sequence typing (MLST). Plasmids were classified based on the DNA sequences of replication initiators. Transposons were detected by PCR. Locations of blaKPC gene were verified through complete sequencing of the plasmids by next-generation sequencing (NGS).@*Results@#A total of 125 strains were collected. K. pneumoniae strains accounted for 88.8% (111) and among them, 103 were ST11 type. IncF plasmids were detected in 48.6% of K. pneumoniae strains and most of them carried mutant Tn1721/Tn4401 chimera (48/54 isolates). Untypable plasmids were discovered in 50.5% of isolated strains and most of them were positive for the wild-type chimera (54/56 isolates). IncF-positive strains isolated during the period of 2011 and 2013 accounted for 94.4%, followed by a dramatic decrease. However, 76.8% of the strains harboring untypable plasmids were isolated from 2014 to 2016 and the number increased year by year.@*Conclusion@#K. pneumoniae of ST11 type was the main cause of blaKPC gene dissemination in Lishui area. Strains carrying the IncF plasmids integrated with the mutant Tn1721/Tn4401 chimera and the untypable plasmids with the wild-type chimera were prevalent before and after 2014, respectively.
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OBJECTIVE To study the methodology of achieving stable co-expression of drug-metab?olizing enzymes in the HepG2 cells by the piggyBac (PB) transposon system. METHODS N-terminal attachment of enhanced green fluorscent protein plasmid (pEGFP- N2) and 2A peptide linked recombinant PB transposon plasmid containing dual-genes encoding drug metabolizing enzymes cyto?chrome P450 3A4 (CYP3A4) and CYP2C19 (pPB-CYP3A4-2A-2C19) were transfected into HepG2 cells respectively by Lipofectamine?LTX reagent, GenJetTM (Ver.Ⅱ) reagent and Neon?Transfection System reagent, which were widely used for large-sized DNA fragments transfection. 48 h later, the transfection efficiency and cell toxicity were detected and compared between the three methods so as to find a method with relatively high efficiency and low toxicity for later transfection.Then,three groups of recombinant PB transposons-single-gene transposon (PB-CYP3A4), 2A peptide linked dual-gene transposon (PB-CYP3A4-2A-2C19) and multiple single-gene transposon mixture〔PB-CYP3A4, PB-CYP2C8, PB-CYP2A6, organic anion transporting polypeptide 1B1 PB transposon (PB-OATP1B1)〕-were transfected into HepG2 cells respectively with the above established method.The puromycin (Puro)-resistant and GFP positive cell clones were picked up and further cultured. The mRNA, protein and metabolic levels of drug-metabolizing enzymes in monoclonal cell lines were detected by quantitative real-time PCR,Western blotting and high performance liquid chromatography-tandem mass spectrometry respectively after screening by Puro and green fluorescence. Comparisons of different groups were made using statistical analysis. RESULTS The comparison of three different transfection methods indi?cated that the transfection efficiency of GenJetTMwas up to(94.2±2.5)% and (89.3±3.3)%,significantly higher than those of the other two methods (P<0.01), along with lower cytotoxicity. Then GenJetTMwas chosen for later transfection. In the Puro-resistant monoclonal cell lines of single transposon PB-CYP3A4,PB-CYP3A4-2A-2C19 groups,the mRNA,protein and enzyme activity levels of drug-metabo?lizing enzymes were significantly increased respectively.The recombinant transposon (PB-CYP3A4-2A-2C19) containing 2A peptide could achieve stable and efficient co-expression of two metabolizing enzymes CYP3A4 and CYP2C19,while the expression of drug-metabolizing enzymes remained unbal?anced and random in those of multiple single-gene transposon mixture group (PB-CYP3A4, PB-CYP2C8,PB-CYP2A6,PB-OATP 1B1)(CYP3A4 was expressed in some cell clones only).CONCLUSION GenJetTM could be an effective method for the PB recombinant transposon transfection into HepG2 cells, by which the PB transposon could mediate stable expression of drug-metabolizing enzymes. In terms of multi-gene expression,a low and unbalanced expression is found by multiple transposons co-transfection method,which is different from that by virus mediated method.In contrast,mono-PB trans?poson linked by 2A peptide can achieve stable expression of multi-genes.
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ABSTRACT The Tc1/Mariner sequence was isolated and mapped on chromosomes aiming to verify the association of this transposable element (TE) and chromosomal rearrangements in Rineloricaria. Cytogenetic analysis showed that Tc1/Mariner does not co-localize with chromosomal fusion points, in addition the analysis revealed intense molecular degeneration in its DNA sequence.
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In this study, the mobilomes of nine teleost species were annotated by bioinformatics methods. Both of the mobilome size and constitute displayed a significant difference in 9 species of teleost fishes. The species of mobilome content ranking from high to low were zebrafish, medaka, tilapia, coelacanth, platyfish, cod, stickleback, tetradon and fugu. Mobilome content and genome size were positively correlated. The DNA transposons displayed higher diversity and larger variation in teleost (0.50% to 38.37%), was a major determinant of differences in teleost mobilomes, and hAT and Tc/Mariner superfamily were the major DNA transposons in teleost. RNA transposons also exhibited high diversity in teleost, LINE transposons accounted for 0.53% to 5.75% teleost genomic sequences, and 14 superfamilies were detected. L1, L2, RTE and Rex retrotransposons obtained significant amplification. While LTR displayed low amplification in most teleost with less than 2% of genome coverages, except in zebrafish and stickleback, where LTR reachs 5.58% and 2.51% of genome coverages respectively. And 6 LTR superfamilies (Copia, DIRS, ERV, Gypsy, Ngaro and Pao) were detected in the teleost, and Gypsy exhibits obvious amplication among them. While the SINE represents the weakest ampification types in teleost, only within zebrafish and coelacanth, it represents 3.28% and 5.64% of genome coverages, in the other 7 teleost, it occupies less than 1% of genomes, and tRNA, 5S and MIR families of SINE have a certain degree of amplification in some teleosts. This study shows that the teleost display high diversity and large variation of mobilome, there is a strong correlation with the size variations of genomes and mobilome contents in teleost, mobilome is an important factor in determining the teleost genome size.
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Miniature inverted-repeat transposable elements transposon is a special transposon that could transpose by "cut-paste" mechanism, which is one of characteristics of DNA transposons. Otherwise, the copy number of MITEs is very high, which is one of characteristics of RNA transposons. Many MITE families have been reported, but little about active MITEs. We summarize recent advances in studying active MITEs. Most the MITEs belong to the Tourist-like family, such as mPing, mGing, PhTourist1, Tmi1 and PhTst-3. Additionally, DTstu1 and MITE-39 belong to Stowaway-like family, and AhMITEs1 belongs to Mutator-like family. Moreover, we summarize the structure (terminal inverse repeats and target site duplications), copy number, evolution pattern and transposition characteristics of these active MITEs, to provide the foundation for the identification of other active MITEs and subsequent research on MITE transposition and amplification mechanism.
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DNA transposon is a kind of factor that is able to translocate gene in its genome, thus offering an efficient method for permanently modifying the genome of mammals. The piggyBac (PB) transposon system has been proven effective in mammalian genomic engineering, including cancer gene discovery, animal transgenesis, in vivo gene delivery as well as in vitro genetic modification like induced pluripotent stem cells. In addition, piggyBac has many desirable features, such as seamless excision of transposons from the genomic DNA and the potential to target integration events to desired DNA sequences. Therefore, the piggyBac translocation system is an ideal genetic tool in the construction of animal models and gene therapy, and we can anticipate that the piggyBac transposon system will play a more and more important role in biomedical research.
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Objective@#An innovative technique was established to rapidly construct various cell lines that could be induced to express multiple influenza A virus (IAV) proteins.@*Method@#The HA protein genes of multiple IAVs were cloned into the Cumate-induced expression system which was positioned between two PiggyBac transposon sites. These HA plasmids were transfected into the HEK293A cell line with the PiggyBac transposase plasmids. The transfected cells were screened with puromycin, and after that the corresponding virus proteins were induced with Cumate.@*Results@#The results of flow cytometry and Western blotting showed that the virus proteins were expressed in most of the cells in corresponding lines after the induction of Cumate.@*Conclusion@#Cell lines which were inducible to express IVA HA protein can be efficiently constructed by using the PiggyBac transposon system.