稳定表达Cas9蛋白的SW620细胞株构建

摘要 目的：建立稳定表达Cas9蛋白的SW620人结肠癌细胞系的单克隆细胞株,提高基因编辑效率,为利用基于CRISPR/Cas9技术的高通量筛选结肠癌相关致病基因提供细胞工具。方法：用Cas9慢病毒侵染SW620细胞系,用致死剂量的puro筛选5-7天,通过有限稀释法获得单克隆细胞株。提取单克隆细胞基因组进行Sanger测序,筛选出含有Cas9基因序列的单克隆细胞株。利用基于SSA修复荧光素酶的报告系统检测单克隆细胞株中Cas9的编辑活性,并通过细胞增殖实验检测Cas9蛋白的表达是否影响细胞增殖。结果：获得了两个表达Cas9蛋白的SW620单克隆细胞株,并通过Sanger测序验证了Cas9序列;荧光素酶报告系统检测显示单克隆细胞株的Cas9蛋白有较高的编辑活性;细胞增殖实验显示Cas9蛋白的表达对SW620增殖活性影响不大。结论：本研究利用慢病毒感染的方式,构建了稳定表达Cas9蛋白的SW620单克隆细胞株,为后续大规模筛选与人结肠癌相关的基因突变提供了细胞工具。     

Generation of a stable mammalian cell line for simultaneous expression of multiple genes by using 2A peptide-based lentiviral vector

Generation of mammalian cells stably expressing multiple exogenous genes is currently difficult. Here we provide a strategy to facilitate this process. First, a helper vector p2A containing three coding sequences for viral 2A peptides was constructed. Three reporter genes coding for red fluorescent protein (DsRed), firefly luciferase (Fluc) and enhanced green fluorescent protein (EGFP) were then inserted into p2A to form a fusion open reading frame that was subsequently subcloned into a lentiviral vector. After transduction, EGFP-positive 293T cells were selected by fluorescence activated cell sorting. The expression of exogenous genes in selected cells was stable for more than 15 passages, and EGFP-positive cells were over 95%. The efficient cleavages of 2A-peptide mediated polyprotein were also observed and all three reporter proteins were functional. Thus, a stable DsRed/Fluc/EGFP-coexpressing cell line was readily established within a short time. The strategy could be useful for basic research and protein production. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Versatile Dual Reporter Gene Systems for Investigating Stop Codon Readthrough in Plants

Background

Translation is most often terminated when a ribosome encounters the first in-frame stop codon (UAA, UAG or UGA) in an mRNA. However, many viruses (and some cellular mRNAs) contain “stop” codons that cause a proportion of ribosomes to terminate and others to incorporate an amino acid and continue to synthesize a “readthrough”, or C-terminally extended, protein. This dynamic redefinition of codon meaning is dependent on specific sequence context.

Methodology

We describe two versatile dual reporter systems which facilitate investigation of stop codon readthrough in vivo in intact plants, and identification of the amino acid incorporated at the decoded stop codon. The first is based on the reporter enzymes NAN and GUS for which sensitive fluorogenic and histochemical substrates are available; the second on GST and GFP.

Conclusions

We show that the NAN-GUS system can be used for direct in planta measurements of readthrough efficiency following transient expression of reporter constructs in leaves, and moreover, that the system is sufficiently sensitive to permit measurement of readthrough in stably transformed plants. We further show that the GST-GFP system can be used to affinity purify readthrough products for mass spectrometric analysis and provide the first definitive evidence that tyrosine alone is specified in vivo by a ‘leaky’ UAG codon, and tyrosine and tryptophan, respectively, at decoded UAA, and UGA codons in the Tobacco mosaic virus (TMV) readthrough context.     

A functional selection of viral genetic elements in cultured cells to identify hepatitis C virus RNA translation inhibitors

We developed a functional selection system based on randomized genetic elements (GE) to identify potential regulators of hepatitis C virus (HCV) RNA translation, a process initiated by an internal ribosomal entry site (IRES). A retroviral HCV GE library was introduced into HepG2 cells, stably expressing the Herpes simplex virus thymidine kinase (HSV-TK) under the control of the HCV IRES. Cells that expressed transduced GEs inhibiting HSV-TK were selected via their resistance to ganciclovir. Six major GEs were rescued by PCR on the selected cell DNA and identified as HCV elements. We validated our strategy by further studying the activity of one of them, GE4, encoding the 5′ end of the viral NS5A gene. GE4 inhibited HCV IRES-, but not cap-dependent, reporter translation in human hepatic cell lines and inhibited HCV infection at a post-entry step, decreasing by 85% the number of viral RNA copies. This method can be applied to the identification of gene expression regulators.     

Targeted Genomic Integration of a Selectable Floxed Dual Fluorescence Reporter in Human Embryonic Stem Cells

The differentiation of pluripotent stem cells involves transition through a series of specific cell states. To understand these cell fate decisions, the field needs improved genetic tools for the labeling, lineage tracing and selection of specific cell types from heterogeneous differentiating populations, particularly in the human embryonic stem cell (hESC) system. We used zinc finger nuclease technology to stably insert a unique, selectable, floxed dual-fluorescence reporter transgene into the AAVS1 locus of RUES2 hESCs. This “stoplight” transgene, mTmG-2a-Puro, strongly expresses membrane-localized tdTomato red fluorescent protein until Cre-dependent recombination causes a switch to expression of membrane-localized enhanced green fluorescent protein (eGFP) and puromycin resistance. First, to validate this system in undifferentiated cells, we transduced transgenic hESCs with a lentiviral vector driving constitutive expression of Cre and observed the expected phenotypic switch. Next, to demonstrate its utility in lineage-specific selection, we transduced differentiated cultures with a lentiviral vector in which the striated muscle-specific CK7 promoter drives Cre expression. This yielded near-homogenous populations of eGFP⁺ hESC-derived cardiomyocytes. The mTmg-2a-Puro hESC line described here represents a useful new tool for both in vitro fate mapping studies and the selection of useful differentiated cell types.     

Proteomic analysis of reporter genes for molecular imaging of transplanted embryonic stem cells

Study of stem cells may reveal promising treatment for diseases. The fate and function of transplanted stem cells remain poorly defined. Recent studies demonstrate that reporter genes can monitor real-time survival of transplanted stem cells in living subjects. We examined the effects of a novel and versatile triple fusion (TF) reporter gene construction on embryonic stem (ES) cell function by proteomic analysis. Murine ES cells were stably transduced with a self-inactivating lentiviral vector containing fluorescence (firefly luciferase; Fluc), bioluminescence (monomeric red fluorescence protein; mRFP), and positron emission tomography (herpes simplex virus type 1 truncated thymidine kinase; tTK) reporter genes. Fluorescence-activated cell sorting (FACS) analysis isolated stably transduced populations. TF reporter gene effects on cellular function were evaluated by quantitative proteomic profiling of control ES cells versus ES cells stably expressing the TF construct (ES-TF). Overall, no significant changes in protein quantity were observed. TF reporter gene expression had no effect on ES cell viability, proliferation, and differentiation capability. Molecular imaging studies tracked ES-TF cell survival and proliferation in living animals. In summary, this is the first proteomic study, demonstrating the unique potential of reporter gene imaging for tracking ES cell transplantation non-invasively, repetitively, and quantitatively.     

Small molecule Y-320 stimulates ribosome biogenesis,protein synthesis,and aminoglycoside-induced premature termination codon readthrough

Premature termination codons (PTC) cause over 10% of genetic disease cases. Some aminoglycosides that bind to the ribosome decoding center can induce PTC readthrough and restore low levels of full-length functional proteins. However, concomitant inhibition of protein synthesis limits the extent of PTC readthrough that can be achieved by aminoglycosides like G418. Using a cell-based screen, we identified a small molecule, the phenylpyrazoleanilide Y-320, that potently enhances TP53, DMD, and COL17A1 PTC readthrough by G418. Unexpectedly, Y-320 increased cellular protein levels and protein synthesis, measured by SYPRO Ruby protein staining and puromycin labeling, as well as ribosome biogenesis measured using antibodies to rRNA and ribosomal protein S6. Y-320 did not increase the rate of translation elongation and it exerted its effects independently of mTOR signaling. At the single cell level, exposure to Y-320 and G418 increased ribosome content and protein synthesis which correlated strongly with PTC readthrough. As a single agent, Y-320 did not affect translation fidelity measured using a luciferase reporter gene but it enhanced misincorporation by G418. RNA-seq data showed that Y-320 up-regulated the expression of CXC chemokines CXCL10, CXCL8, CXCL2, CXCL11, CXCL3, CXCL1, and CXCL16. Several of these chemokines exert their cellular effects through the receptor CXCR2 and the CXCR2 antagonist SB225002 reduced cellular protein levels and PTC readthrough in cells exposed to Y-320 and G418. These data show that the self-limiting nature of PTC readthrough by G418 can be compensated by Y-320, a potent enhancer of PTC readthrough that increases ribosome biogenesis and protein synthesis. They also support a model whereby increased PTC readthrough is enabled by increased protein synthesis mediated by an autocrine chemokine signaling pathway. The findings also raise the possibility that inflammatory processes affect cellular propensity to readthrough agents and that immunomodulatory drugs like Y-320 might find application in PTC readthrough therapy.

A cell-based screen identifies a small molecule, Y-320, that potently enhances readthrough of premature termination codons by the aminoglycoside G418. Y-320 increases cellular protein levels and protein synthesis, and appears to act via an autocrine chemokine signaling pathway.     

慢病毒介导的稳定表达人GM-CSF、IL-4的EA.hy926细胞株的建立

目的:获得能够稳定表达人粒细胞-巨噬细胞集落刺激因子(GM-CSF)和白细胞介素4(IL-4)的内皮细胞株。方法:构建重组慢病毒表达载体,在293FT细胞中进行病毒的包装,用获得的高滴度慢病毒感染EA.hy926细胞株,建立能够稳定、高效表达hGM-CSF、hIL-4的EA.hy926细胞株。结果:EA.hy926感染效率达90%以上,长期传代后阳性率仍维持在90%以上;实时定量PCR和ELISA方法证实感染后细胞hGM-CSF、hIL-4在mRNA水平的表达分别是未感染细胞的25倍和9946倍,在蛋白水平的表达分别是未感染细胞的145倍和23倍。结论:构建的细胞系可以稳定表达细胞因子GM-CSF、IL-4。     

New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae

Stop codon readthrough may be promoted by the nucleotide environment or drugs. In such cases, ribosomes incorporate a natural suppressor tRNA at the stop codon, leading to the continuation of translation in the same reading frame until the next stop codon and resulting in the expression of a protein with a new potential function. However, the identity of the natural suppressor tRNAs involved in stop codon readthrough remains unclear, precluding identification of the amino acids incorporated at the stop position. We established an in vivo reporter system for identifying the amino acids incorporated at the stop codon, by mass spectrometry in the yeast Saccharomyces cerevisiae. We found that glutamine, tyrosine and lysine were inserted at UAA and UAG codons, whereas tryptophan, cysteine and arginine were inserted at UGA codon. The 5′ nucleotide context of the stop codon had no impact on the identity or proportion of amino acids incorporated by readthrough. We also found that two different glutamine tRNA^Gln were used to insert glutamine at UAA and UAG codons. This work constitutes the first systematic analysis of the amino acids incorporated at stop codons, providing important new insights into the decoding rules used by the ribosome to read the genetic code.     

Dual Transgene Expression in Murine Cerebellar Purkinje Neurons by Viral Transduction In Vivo

Viral-vector mediated gene transfer to cerebellar Purkinje neurons in vivo is a promising avenue for gene therapy of cerebellar ataxias and for genetic manipulation in functional studies of animal models of cerebellar disease. Here, we report the results of experiments designed to identify efficient methods for viral transduction of adult murine Purkinje neurons in vivo. For these analyses, several lentiviral and an adeno-associated virus (AAV), serotype 1, vector with various promoter combinations were generated and compared for in situ transduction efficiency, assayed by fluorescent reporter protein expression in Purkinje neurons. Additional experiments were also conducted to identify the optimal experimental strategy for co-expression of two proteins in individual Purkinje neurons. Of the viruses tested, AAV1 with a CAG promoter exhibited the highest specificity for Purkinje neurons. To deliver two proteins to the same Purkinje neuron, several methods were tested, including: an internal ribosome entry site (IRES), a 2A sequence, a dual promoter vector, and co-injection of two viruses. Efficient expression of both proteins in the same Purkinje neuron was only achieved by co-injecting two AAV1-CAG viruses. We found that use of an AAV1-CAG virus outperformed similar lentivirus vectors and that co-injection of two AAV1-CAG viruses could be used to efficiently deliver two proteins to the same Purkinje neuron in adult mice. AAV1 with a CAG promoter is highly efficient and selective at transducing adult cerebellar Purkinje neurons and two AAV-CAG viruses can be used to efficiently express two proteins in the same neuron in vivo.     

HDAC2 sumo-E3连接酶活性在DLD1细胞迁移和增殖中的作用

目的:探讨HDAC2 sumo-E3连接酶对DLD1细胞迁移与增殖的影响和可能机制。方法:全基因合成HDAC2 sumo-E3连接酶突变体HDAC2(325-488),并构建其重组慢病毒表达载体;感染DLD1hdac2-/-细胞(无内源性HDAC2表达),筛选稳定表HDAC2(325-488)的细胞克隆DLD1h325-488;RTCA实时细胞分析仪、Trans-well等检测DLD1h325-488稳定细胞的增殖和迁移能力;免疫印迹、q RT-PCR检测DLD1h325-488稳定细胞增殖和迁移相关基因的表达。结果:构建了稳定表达HDAC2 sumo-E3连接酶突变体HDAC2(325-488)的细胞株DLD1h325-488,并获得稳定表达HDAC2 sumo-E3连接酶突变体HDAC2(325-488)的DLD1h325-488细胞。与对照组细胞相比,DLD1h325-488细胞增殖活性无显著变化,但是细胞的迁移能力和迁移相关蛋白MMP14的表达显著上升。结论:HDAC2 sumo-E3连接酶可能通过上调mmp14的表达而促进DLD1细胞的迁移。     

Discrimination between defects in elongation fidelity and termination efficiency provides mechanistic insights into translational readthrough

The suppression of stop codons (termed translational readthrough) can be caused by a decreased accuracy of translation elongation or a reduced efficiency of translation termination. In previous studies, the inability to determine the extent to which each of these distinct processes contributes to a readthrough phenotype has limited our ability to evaluate how defects in the translational machinery influence the overall termination process. Here, we describe the combined use of misincorporation and readthrough reporter systems to determine which of these mechanisms contributes to translational readthrough in Saccharomyces cerevisiae. The misincorporation reporter system was generated by introducing a series of near-cognate mutations into functionally important residues in the firefly luciferase gene. These constructs allowed us to monitor the incidence of elongation errors by monitoring the level of firefly luciferase activity from a mutant allele inactivated by a single missense mutation. In this system, an increase in luciferase activity should reflect an increased level of misincorporation of the wild-type amino acid that provides an estimate of the overall fidelity of translation elongation. Surprisingly, we found that growth in the presence of paromomycin stimulated luciferase activity for only a small subset of the mutant proteins examined. This suggests that the ability of this aminoglycoside to induce elongation errors is limited to a subset of near-cognate mismatches. We also found that a similar bias in near-cognate misreading could be induced by the expression of a mutant form of ribosomal protein (r-protein) S9B or by depletion of r-protein L12. We used this misincorporation reporter in conjunction with a readthrough reporter system to show that alterations at different regions of the ribosome influence elongation fidelity and termination efficiency to different extents.     

Human mesenchymal stem cells (hMSCs) expressing truncated soluble vascular endothelial growth factor receptor (tsFlk-1) following lentiviral-mediated gene transfer inhibit growth of Burkitt's lymphoma in a murine model

BACKGROUND: Efficient gene transfer to bone marrow derived mesenchymal stem cells (MSC) would provide an important opportunity to express potent anticancer agents in the tumour microenvironment because of their contribution to the tumour stroma. METHODS: HIV-based lentiviral vectors were pseudotyped with four different envelope proteins; amphotropic murine leukaemia virus (ampho), murine leukaemia virus (10A1), feline endogenous virus (RD114), and the vesicular stomatitis virus glycoprotein (VSVG). These pseudotypes were examined for transduction efficiency in human bone marrow derived MSC. The effect of lentiviral expression of truncated soluble vascular endothelial growth factor decoy receptor (tsFlk-1) in MSC on growth of Raji cells was determined, both in vitro and in vivo. RESULTS: All lentiviral vectors produced significant levels of transduction at an multiplicity of infection (MOI) of 1, those bearing the RD114 envelope glycoprotein consistently produced higher transduction levels (mean 70 +/- 6%) compared with the other pseudotyped lentiviral vectors, although there was significant inter-donor variation. Stable transgene expression was achieved after multiple rounds of transduction with VSVG-pseudotyped particles, without alteration in the differentiative capacity of transduced cells. Co-injection of MSC stably expressing tsFlk-1 with Raji Burkitt's lymphoma cells significantly impaired subcutaneous tumour growth in immunodeficient mice when compared to controls where either unmanipulated MSC or GFP-expressing MSC were used. CONCLUSIONS: Human MSC are easily transduced by pseudotyped lentiviral particles but there is inter-donor variation in transduction efficiency. Gene-modified MSC expressing a gene of therapeutic potential can moderate growth of haematological malignancies.     

Evidence of efficient stop codon readthrough in four mammalian genes

Stop codon readthrough is used extensively by viruses to expand their gene expression. Until recent discoveries in Drosophila, only a very limited number of readthrough cases in chromosomal genes had been reported. Analysis of conserved protein coding signatures that extend beyond annotated stop codons identified potential stop codon readthrough of four mammalian genes. Here we use a modified targeted bioinformatic approach to identify a further three mammalian readthrough candidates. All seven genes were tested experimentally using reporter constructs transfected into HEK-293T cells. Four displayed efficient stop codon readthrough, and these have UGA immediately followed by CUAG. Comparative genomic analysis revealed that in the four readthrough candidates containing UGA-CUAG, this motif is conserved not only in mammals but throughout vertebrates with the first six of the seven nucleotides being universally conserved. The importance of the CUAG motif was confirmed using a systematic mutagenesis approach. One gene, OPRL1, encoding an opiate receptor, displayed extremely efficient levels of readthrough (∼31%) in HEK-293T cells. Signals both 5′ and 3′ of the OPRL1 stop codon contribute to this high level of readthrough. The sequence UGA-CUA alone can support 1.5% readthrough, underlying its importance.     

A novel stop codon readthrough mechanism produces functional Headcase protein in Drosophila trachea

Translational regulation provides an efficient means to control the localization and production of proteins. The headcase (hdc) mRNA in Drosophila generates two overlapping proteins as a result of translational readthrough of an internal UAA stop codon. This readthrough event is necessary for the function of hdc as a branching inhibitor during tracheal development. By ectopic expression of different Hdc proteins in the trachea, we show that the long Hdc form alone, can function as a potent branching inhibitor whose activity is proportional to its amount. The suppression of termination in the hdc mRNA is not stop-codon dependent, suggesting that the readthrough does not involve codon specific suppressors. We have identified an 80 nucleotide sequence immediately downstream of the UAA, which is necessary and sufficient to confer termination readthrough in a heterologous mRNA. We present a novel mechanism of eukaryotic translational termination suppression that may regulate the amount of functional Hdc.     

Codon reassignment to facilitate genetic engineering and biocontainment in the chloroplast of Chlamydomonas reinhardtii

There is a growing interest in the use of microalgae as low‐cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein‐coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome‐binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co‐introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae.     

Establishment of a novel immortalized human prostatic epithelial cell line stably expressing androgen receptor and its application for the functional screening of androgen receptor modulators

In this study, we developed a human prostatic epithelial cell line BPH-1-AR stably expressing AR by lentiviral transduction. Characterization by immunoblot and RT-PCR showed that AR was stably expressed in all representative BPH-1-AR clones. Androgen treatment induced a secretory differentiation phenotype in BPH-1-AR cells but suppressed their cell proliferation. Treatments with AR agonists induced transactivation of a transfected PSA-gene promoter reporter in BPH-1-AR cells, whereas this transactivation was suppressed by an AR antagonist flutamide, indicating that the transduced AR in BPH-1-AR cells was functional. Finally, we utilized BPH-1-AR cells to evaluate the androgenic activities and growth effects of five newly developed non-steroidal compounds. Results showed that these compounds showed androgenic activities and growth-inhibitory effects on BPH-1-AR cells. Our results showed that BPH-1-AR cell line would be a valuable in vitro model for the study of androgen-regulated processes in prostatic epithelial cells and identification of compounds with AR-modulating activities.