Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES.     

Strategy for increased efficiency of transfection in human cell lines using radio frequency electroporation

Traditional electroporation devices use direct current electric fields to stimulate the uptake of oligonucleotides, plasmids, short peptides, and proteins into a variety of cell types. A variation of this widely used technique is now available which relies on radio frequency (RF) electrical pulses. This oscillating type of electrical field reportedly elicits greater uptake of plasmid DNA across the plasma membrane. We evaluated a protocol for RF electroporation of the a human embryonic kidney cell line and a Burkitt's lymphoma (BL) cell line for effeciency of transfection by RF electroporation. The plasmid EGFP, which codes for the widely used fusion protein, enhanced green fluorescent protein (EGFP), was used as a reporter of plasmid uptake after transfections. Transfection efficiency consistently increased approximately 30% from that typically obtained with conventional DC type electroporation and was accompanied by greater survivability of cells. Additionally, in some instances, percent transfection efficiency increased to over 70%. Thus, RF electroporation represents an improved methodology for transfection of human cell lines. Moreover, the RF protocol is simple to incorporate in laboratories already utilizing conventional electroporation devices and techniques.     

A one-step cloning method for the construction of somatic cell gene targeting vectors: application to production of human knockout cell lines

Background

To facilitate the screening of large quantities of new antimicrobial peptides (AMPs), we describe a cost-effective method for high throughput prokaryotic expression of AMPs. EDDIE, an autoproteolytic mutant of the N-terminal autoprotease, Npro, from classical swine fever virus, was selected as a fusion protein partner. The expression system was used for high-level expression of six antimicrobial peptides with different sizes: Bombinin-like peptide 7, Temporin G, hexapeptide, Combi-1, human Histatin 9, and human Histatin 6. These expressed AMPs were purified and evaluated for antimicrobial activity.

Results

Two or four primers were used to synthesize each AMP gene in a single step PCR. Each synthetic gene was then cloned into the pET30a/His-EDDIE-GFP vector via an in vivo recombination strategy. Each AMP was then expressed as an Npro fusion protein in Escherichia coli. The expressed fusion proteins existed as inclusion bodies in the cytoplasm and the expression levels of the six AMPs reached up to 40% of the total cell protein content. On in vitro refolding, the fusion AMPs was released from the C-terminal end of the autoprotease by self-cleavage, leaving AMPs with an authentic N terminus. The released fusion partner was easily purified by Ni-NTA chromatography. All recombinant AMPs displayed expected antimicrobial activity against E. coli, Micrococcus luteus and S. cerevisia.

Conclusions

The method described in this report allows the fast synthesis of genes that are optimized for over-expression in E. coli and for the production of sufficiently large amounts of peptides for functional and structural characterization. The Npro partner system, without the need for chemical or enzymatic removal of the fusion tag, is a low-cost, efficient way of producing AMPs for characterization. The cloning method, combined with bioinformatic analyses from genome and EST sequence data, will also be useful for screening new AMPs. Plasmid pET30a/His-EDDIE-GFP also provides green/white colony selection for high-throughput recombinant AMP cloning.     

A new strategy for gene targeting and functional proteomics using the DT40 cell line

DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry–based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination–based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.     

A simple analysis system for the estimation of recombination efficiency using fluorescence-activated cell sorting

A simple and accurate analysis system for the estimation of recombination efficiency in vivo using fluorescence-activated cell sorting (FACS) was designed and was subsequently used to compare the efficiency of recombination related to different spacer mutants. F(3) and F(5) mutant sequences were used for Flpe-mediated cassette exchange, and m2 and lox2272 mutant sequences were used for Cre-mediated cassette exchange due to their high incompatibility with wild-type sequences. The incompatibilities with wild-type were almost the same between mutant sequences. However, the recombination efficiencies were different. F(3) and m2 could mediate more efficient recombination than F(5) and lox2272, respectively. These results are consistent with the fact that the sequence of spacer region affects not only the reactivity upon wild-type sequence but also the recombination efficiency. It was also confirmed that the recombination process was mediated in a site-specific manner through PCR analysis using different sizes of exchange cassettes. In this experiment, the feasibility of the FACS analysis system for the estimation of recombination efficiency was verified. This system should be readily applicable for estimating recombination efficiency of other various mutant candidates, which will contribute to more precise and efficient site-specific recombination.     

Essential role for gene profiling analysis in the authentication of human cell lines

Cross-contamination between cultured cell lines can result in the generation of erroneous scientific data. Hence, it is very important to eliminate cell lines that are of an origin different from that being claimed. Inter-species contamination can be detected by various established methods, such as karyotype and isozyme analyses. However, it has been impossible to detect intraspecies cross-contamination prior to the development of technology to detect differences between cell lines at the molecular level. Recently, profiling of short tandem repeat (STR) polymorphisms has been established as a method for the analyses of gene polymorphism. Gene profiling by STR polymorphism (STR profiling) is a simple and reliable method to identify individual cell lines. Each human cell line currently provided by the Cell Engineering Division of the RIKEN BioResource Center was analyzed by STR profiling to authenticate its identity. We found that more than 10 human cell lines out of approximately 400 were in fact identical to a different cell line deposited in the collection, and therefore had been misidentified. We conclude that STR profiling is a useful and powerful method for eliminating cell lines that have been misidentified by cross-contamination or by other causes. Hence, STR profiling of human cell lines used in published research will likely be a prerequisite for publication in the future, so that the problem of misidentification of cell lines can be eliminated.     

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines

Background

Specific and efficient delivery of genes into targeted cells is a priority objective in non‐viral gene therapy. Polyethyleneimine‐based polyplexes have been reported to be good non‐viral transfection reagents. However, polyplex‐mediated DNA delivery occurs through a non‐specific mechanism. This article reports the construction of an immunopolyplex, a targeted non‐viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines.

Methods

Targeting elements (biotin‐labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19?), Granta 519 (CD3?/ CD19+), and J.RT3‐T3.5 (CD3?/CD19?)] using the EGFP gene as a reporter gene and anti‐CD3 and anti‐CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry.

Results

A significant selectivity of gene delivery was observed, since the anti‐CD3 immunopolyplex worked only in Jurkat cells while the anti‐CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3? cell mixture with anti‐CD3 immunopolyplexes showed up to 16‐fold more transfection in CD3+ than in CD3? cells. Several non‐specific transfection reagents showed poor or no transfection activity.

Conclusion

It is concluded that immunopolyplex is a good non‐viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) – the streptavidin–polyplex backbone remaining intact – thereby conferring high versatility. Copyright © 2002 John Wiley & Sons, Ltd.

     

Genetic inheritance of gene expression in human cell lines

Combining genetic inheritance information, for both molecular profiles and complex traits, is a promising strategy not only for detecting quantitative trait loci (QTLs) for complex traits but for understanding which genes, pathways, and biological processes are also under the influence of a given QTL. As a primary step in determining the feasibility of such an approach in humans, we present the largest survey to date, to our knowledge, of the heritability of gene-expression traits in segregating human populations. In particular, we measured expression for 23,499 genes in lymphoblastoid cell lines for members of 15 Centre d'Etude du Polymorphisme Humain (CEPH) families. Of the total set of genes, 2,340 were found to be expressed, of which 31% had significant heritability when a false-discovery rate of 0.05 was used. QTLs were detected for 33 genes on the basis of at least one P value <.000005. Of these, 13 genes possessed a QTL within 5 Mb of their physical location. Hierarchical clustering was performed on the basis of both Pearson correlation of gene expression and genetic correlation. Both reflected biologically relevant activity taking place in the lymphoblastoid cell lines, with greater coherency represented in Kyoto Encyclopedia of Genes and Genomes database (KEGG) pathways than in Gene Ontology database pathways. However, more pathway coherence was observed in KEGG pathways when clustering was based on genetic correlation than when clustering was based on Pearson correlation. As more expression data in segregating populations are generated, viewing clusters or networks based on genetic correlation measures and shared QTLs will offer potentially novel insights into the relationship among genes that may underlie complex traits.     

A REIC gene shows down-regulation in human immortalized cells and human tumor-derived cell lines

Normal human cells stop proliferation after a certain number of cell divisions. This phenomenon is called cellular aging. The fact that the senescence phenotype is dominant and the immortal one is recessive indicates that immortalization of human cells may be caused by loss of functions of certain genes in normal cells. Based on this evidence, several cDNA clones whose expression was down-regulated during the immortalization process of human cells were isolated by the representative difference analysis (RDA) system in our laboratory. One of them, which was named REIC, was expressed to a lower degree in three human immortalized cell lines as compared with their parental normal counterparts. In addition, the expression of REIC was markedly lower in eight human tumor-derived cell lines (Hep3B and HuH-7 hepatocellular carcinomas, HuH-6 Clone 5 hepatoblastoma, HuCCT-1 cholangiocarcinoma, A549 lung cancer, HaCaT immortalized keratinocyte, HeLa cervical carcinoma, and Saos-2 osteosarcoma). In contrast, among the human tissues examined, the heart and brain, which contain a large number of post-mitotic cells, showed the highest expression of REIC. The full-length REIC cDNA revealed that the predicted protein is 350 amino acids in length and possesses coiled-coil tertiary structures in each of the amino- and carboxyl-termini. Furthermore, a search of the protein database revealed a match of this gene product with Dkk-3, which is a novel inhibitor of Wnt oncogene. These results indicate that the REIC cloned by us may function as a tumor suppressor.     

Cancer-testis gene expression profile in human melanoma cell lines

A growing number of evidence indicates that cancer-testis antigens (CTA) can be used as specific targets for immune therapy of malignant melanoma. The aim of this study was to provide a basis for selecting the most suitable CTA by analyzing the mRNA expression profile of genes encoding CTA in melanoma cell lines. We used a real-time quantitative PCR to measure the expression level for the following genes: GAGE1, NY-ESO-1, MAGEA1, PASD, SCP1, SEMG1, SPANXA, SSX1, and PRAME. The objects of study were cell lines mel P, mel Si, mel Mtp, mel Il, mel Hn, mel Ibr, and mel Kor obtained from patients diagnosed with disseminated melanoma. We established that the highest frequency of occurrence and the highest expression level had the following genes: GAGE1, NY-ESO-1, MAGEA1, SCP1, SPANXA, SSX1, and PRAME. Their mRNA translation products can be promising candidates for immunotherapy.     

Upregulation of activin A gene by butyrate in human colon cancer cell lines

Activin A has been reported to play a role in the progression of colorectal cancer. Because dietary fiber protects against colorectal cancer, we hypothesized that butyrate, a fermentation product of dietary fiber, may affect the expression of activin A in colon cancer cells. Semiquantitative RT-PCR demonstrated that the activin A gene was upregulated by sodium butyrate in the human colon cancer cell lines HT-29 and Caco-2 in a concentration- and time-dependent manner. However, the activin A gene did not respond to sodium butyrate in the human normal colonic cell line FHC, rat normal intestinal epithelial cell (IEC) line IEC-6, and the explant of rat colon. Flow cytometry and agarose gel electrophoresis of genomic DNA revealed that cell cycle arrest and apoptosis were induced by sodium butyrate but not exogenous activin A in HT-29 cells, indicating that activin A could not act as an autocrine factor in colon cancer cells. By assuming that activin A promotes colorectal cancer spread as a paracrine factor, our findings suggest that butyrate could act as a tumor promoter in some circumstances.     

Self-assemble gene delivery system for molecular targeting using nucleic acid aptamer

We have developed a novel vector constructed with pDNA, polyethylenimine (PEI), and mucin 1 (MUC1) aptamer for tumor-targeted gene delivery. The MUC1 aptamer and non-specific aptamer were employed to coat the pDNA/PEI complexes electrostatically and stable nanoparticles were formed. The addition of a non-specific aptamer to the pDNA/PEI complex decreased gene expression in the human lung cancer cell line, A549 cells expressing MUC1 regularly. At the same time, the pDNA/PEI/MUC1 aptamer complex showed higher gene expression than pDNA/PEI/non-specific aptamer complex. Furthermore, the pDNA/PEI/MUC1 aptamer complex showed markedly high gene expression in tumor-bearing mice; thus, pDNA/PEI/MUC1 aptamer complexes are useful as a tumor-targeted gene delivery system with high transfection efficiency.     

利用CRISPR/Cas9敲除人源细胞系中LMNA基因的研究

LMNA基因编码A型和C型核纤层蛋白,参与细胞核核膜的组织,影响基因组稳定性并对细胞分化产生影响。人类肿瘤中LMNA表达异常普遍存在,其突变造成多种核纤层蛋白病,如Emery-Dreifuss肌营养不良症(Emery-Dreifussmusculardystrophy,EDMD)、扩张型心肌病(dilatedcardiomyopathy,DCM)和儿童早老症(Hutchinson-Glifordprogeriasyndrome,HGPS)等。为进一步研究LMNA在细胞内的功能,本研究利用CRISPR/Cas9技术对体外培养的293T与HepG2细胞株的LMNA基因进行编辑,获得两株LMNA基因敲除(LMNA KO)的稳定细胞系。与野生型相比,LMNAKO细胞系增殖能力相对减弱,凋亡增加。同时,细胞形态上也发生显著改变,核膜凹凸不平。本研究首次报道了LMNA KO永生细胞系构建和形态研究结果,为后续LMNA基因功能研究和致病突变体研究奠定基础。     

Efficient somatic gene targeting in the lymphoid human cell line DG75

Among the different approaches used to define the function of a protein of interest, alteration and/or deletion of its encoding gene is the most direct strategy. Homologous recombination between the chromosomal gene locus and an appropriately designed targeting vector results in an alteration or knockout of the gene of interest. Homologous recombination is easily performed in yeast or in murine embryonic stem cells, but is cumbersome in more differentiated and diploid somatic cell lines. Here we describe an efficient method for targeting both alleles of a complex human gene locus in DG75 cells, a cell line of lymphoid origin. The experimental approach included a conditional knockout strategy with three genotypic markers, which greatly facilitated the generation and phenotypic identification of targeted recombinant cells. The vector was designed such that it could be reused for two consecutive rounds of recombination to target both alleles. The human DG75 cell line appears similar to the chicken DT40 pre B-cell line, which supports efficient homologous recombination. Therefore, the DG75 cell line is a favorable addition to the limited number of cell lines amenable to gene targeting and should prove useful for studying gene function through targeted gene alteration or deletion in human somatic cells.     

Improved methods for the generation of human gene knockout and knockin cell lines

Recent studies have demonstrated the utility of recombinant adeno-associated viral (rAAV) vectors in the generation of human knockout cell lines. The efficiency with which such cell lines can be generated using rAAV, in comparison with more extensively described plasmid-based approaches, has not been directly tested. In this report, we demonstrate that targeting constructs delivered by rAAV vectors were nearly 25-fold more efficient than transfected plasmids that target the same exon. In addition, we describe a novel vector configuration which we term the synthetic exon promoter trap (SEPT). This targeting element further improved the efficiency of knockout generation and uniquely facilitated the generation of knockin alterations. An rAAV-based SEPT targeting construct was used to transfer a mutant CTNNB1 allele, encoding an oncogenic form of β-catenin, from one cell line to another. This versatile method was thus shown to facilitate the efficient integration of small, defined sequence alterations into the chromosomes of cultured human cells.     

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.