EGFP Cassette Research Articles

An Enhanced Retroviral Vector for Efficient Genetic Manipulation and Selection in Mammalian Cells.

Introducing genetic material into hard-to-transfect mammalian cell lines and primary cells is often best achieved through retroviral infection. An ideal retroviral vector should offer a compact, selectable, and screenable marker while maximizing transgene delivery capacity. However, a previously published retroviral vector featuring an EGFP/Puromycin fusion protein failed to meet these criteria in our experiments. We encountered issues such as low infection efficiency, weak EGFP fluorescence, and selection against infected cells. To address these shortcomings, we developed a novel retroviral vector based on the Moloney murine leukemia virus. This vector includes a compact bifunctional EGFP and Puromycin resistance cassette connected by a 2A peptide. Our extensively tested vector demonstrated superior EGFP expression, efficient Puromycin selection, and no growth penalty in infected cells compared with the earlier design. These benefits were consistent across multiple mammalian cell types, underscoring the versatility of our vector. In summary, our enhanced retroviral vector offers a robust solution for efficient infection, reliable detection, and effective selection in mammalian cells. Its improved performance and compact design make it an ideal choice for a wide range of applications involving precise genetic manipulation and characterization in cell-based studies.

CRISPR/Cas9-mediated knock-in of a fluorescent reporter into the target locus of interest in human pluripotent stem cells

The method presented herein is associated with the Lab Resource article titled “Generation of αMHC-EGFP knock-in in human pluripotent stem cell line, SNUe003-A-3, using CRISPR/CAS9-based gene targeting” [1]. The cardiac muscle-specific protein, α-myosin heavy chain (αMHC), is encoded by the human MYH6 gene, which is expressed in both the atria and ventricles during embryonic development and is predominantly expressed in the atria after birth [2]. Herein, the methods used to achieve CRISPR/SpCas9-mediated introduction of an EGFP reporter into αMHC, the target locus in human pluripotent stem cells (hPSCs) for cardiac lineage tracing and clinical cell sorting are described. The CRISPR-Cas9 system enables efficient replacement of the stop codon in the last exon of αMHC with a 2A non-joining peptide (T2A)-EGFP cassette. First, hPSCs are transfected with the donor construct and Cas9/sgRNA plasmids via electroporation and selected with neomycin for approximately 3 weeks. Thereafter, the established cell line exhibits typical characteristics of human embryonic stem cells (hESCs). When these cells differentiate into cardiomyocytes, the expression of EGFP is confirmed using confocal microscopy, flow cytometry analysis, and immunostaining.•The line enables monitoring of cell maturation events during human cardiac development.•The line is a valuable platform for cardiotoxicity tests and drug screening.•This method has already been employed in two original studies, as previously reported for reporter cell line generation using CRISPR/Cas9.

TALEN-mediated intron editing of HSPCs enables transgene expression restricted to the myeloid lineage

Gene therapy in hematopoietic stem and progenitor cells (HSPCs) shows great potential for the treatment of inborn metabolic diseases. Typical HSPC gene therapy approaches rely on constitutive promoters to express a therapeutic transgene, which is associated with multiple disadvantages. Here, we propose a novel promoter-less intronic gene editing approach that triggers transgene expression only after cellular differentiation into the myeloid lineage. We integrated a splicing-competent eGFP cassette into the first intron of CD11b and observed expression of eGFP in the myeloid lineage but minimal to no expression in HSPCs or differentiated non-myeloid lineages. In vivo, edited HSPCs successfully engrafted in immunodeficient mice and displayed transgene expression in the myeloid compartment of multiple tissues. Using the same approach, we expressed alpha-L-iduronidase (IDUA), the defective enzyme in Mucopolysaccharidosis type I, and observed a 10-fold supraendogenous IDUA expression exclusively after myeloid differentiation. Edited cells efficiently populated bone marrow, blood, and spleen of immunodeficient mice, and retained the capacity to secrete IDUA ex vivo. Importantly, cells edited with the eGFP and IDUA transgenes were also found in the brain. This approach may unlock new therapeutic strategies for inborn metabolic and neurological diseases that require the delivery of therapeutics in brain.

Monitoring peroxisome dynamics using enhanced green fluorescent protein labeling in Alternaria alternata.

Brown leaf spot on tobacco is a serious fungal disease caused by Alternaria alternata. Peroxisomes are organelles playing an important role in the development and infection of plant pathogenic fungi. But, until now, there is no report on the peroxisome dynamics during the conidia germination of A. alternata. To evaluate the roles of peroxisome in the development of the fungus, in the present work, an enhanced green fluorescent protein (eGFP) cassette tagged with peroxisome targeting signal 2 (PTS2) was integrated into A. alternata to label the organelles, and an eGFP cassette carrying a nuclear located signal (NLS) was performed parallelly. The transformants containing the fusions emitted fluorescence in punctate patterns. The fluorescence of eGFP-PTS2 was distributed exactly in the peroxisomes while those of eGFP-NLS were located in the nucleus. Typical AaGB transformants were selected to be investigated for the peroxisome dynamics. The results showed that during spore germination, the number of peroxisomes in the spores decreased gradually, but increased in the germ tubes. In addition, when the transformants were cultured on lipid media, the numbers of peroxisomes increased significantly, and in a larger portion, present in striped shapes. These findings give some clues for understanding the peroxisomal functions in the development of A. alternata.

Abstract PO-038: LAMC2: New player in stemness and tumor progression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating and essentially incurable disease, typically characterized by high chemoresistance and metastatic spread attributable to cancer stem cells (CSCs). This subpopulation is critical for tumor initiation and recurrence, but the mechanism through which they acquire metastatic traits is not well understood. Hence, targeting the CSC niche and their plasticity could be a complementary therapeutic strategy against cancer. Laminin subunit-γ-2 (LAMC2) is an epithelial basement membrane protein, which controls cell motility and adhesion and is widely expressed in the majority of human tumors. However, its role in PDAC remains largely unknown. In several patient cohorts we observed that high levels of LAMC2 significantly correlated with shorter overall survival. In addition, the tissue microarray analysis on PDAC sections revealed prognostic significance of LAMC2 expression in tumor with high grade of aggressiveness (i.e., G2 and G3). To determine the role of LAMC2 in sustaining tumorigenicity, we knocked down it in patient-derived xenografts (PDX) cells using lentiviral shRNA constructs. The silencing of LAMC2 resulted in decreased self-renewal, invasiveness, tumorigenicity and gemcitabine resistance both in vitro and in vivo. To identify or track the LAMC2 tumor cell population in an intact environment we engineered primary PDAC cells that carry EGFP cassette knocked in the LAMC2 locus through the CRISPR-Cas9 technique. Analysis of LAMC2-EGFP+ cells isolated from tumor demonstrated that these cells express a gene program similar to that of highly metastatic stem cells and that they initiate and propagate both the primary tumor and the metastasis to recipient mice very efficiently compared to their counterpart. In conclusion, we identified a highly metastatic subpopulation of cancer stem cells, characterized by high levels of LAMC2. Strategies aimed at targeting the LAMC2 population may be effective in reducing tumor aggressiveness in combination with conventional therapy. Citation Format: Donatella Delle Cave, Tea Teresa Iavazzo, Maria Mangini, Gennaro Andolfi, Teresa Pirozzi, Annalisa Di Domenico, Annachiara De Luca, Enza Lonardo. LAMC2: New player in stemness and tumor progression in pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-038.

Construction and Characterization of a Novel Bacmid AcBac-Syn Based on a Synthesized Baculovirus Genome

Baculoviruses are large DNA viruses which have been widely used as expression vectors and biological insecticides. Homologous recombination and Bac-to-Bac system have been the main methods for manipulating the baculovirus genome. Recently, we generated a synthetic baculovirus AcMNPV-WIV-Syn1 which fully resembled its parental virus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). Here, we report the modification of AcMNPV-WIV-Syn1 into a novel bacmid, AcBac-Syn, which can be used as a backbone for Bac-to-Bac system. To achieve this, a vector contained a LacZ:attTn7 and egfp cassette was constructed, and recombined with a linearized AcMNPV-WIV-Syn1 genome by transformation-associated recombination in yeast to generate bacmid AcBac-Syn. The bacmid was then transfected to insect cells and the rescued virus showed similar biological characteristics to the wild-type virus in terms of the kinetics of budded virus production, the morphology of occlusion bodies, and the oral infectivity in insect larvae. For demonstration, a red fluorescent protein gene Dsred was transposed into the attTn7 site by conventional Bac-to-Bac method, and the transfection and infection assays showed that AcBac-Syn can be readily used for foreign gene insertion and expression. AcBac-Syn has several advantages over the conventional AcMNPV bacmids, such as it contains an egfp reporter gene which facilitates visualization of virus propagation and titration; its DNA copy numbers could be induced to a higher level in E. coli; and the retaining of the native polyhedrin gene in the genome making it an attractive system for studying the functions of gene related to occlusion body assembly and oral infection.

Optimized Adeno-Associated Virus Vectors for Efficient Transduction of Human Retinal Organoids.

The most widely used vectors for gene delivery in the retina are recombinant adeno-associated virus (rAAV) vectors. They have proven to be safe and effective in retinal gene therapy studies aimed to treat inherited retinal dystrophies, although with various limitations in transduction efficiency. Novel variants with modified capsid sequences have been engineered to improve transduction and overcome limitations of naturally occurring variants. Although preclinical evaluation of rAAV vectors based on such novel capsids is mostly done in animal models, the use of human induced pluripotent stem cell (hiPSC)-derived organoids offers an accessible and abundant human testing platform for rAAV evaluation. In this study, we tested the novel capsids, AAV9.GL and AAV9.NN, for their tropism and transduction efficiency in hiPSC-derived human retinal organoids (HROs) with all major neuronal and glial cell types in a laminated structure. These variants are based on the AAV9 capsid and were engineered to display specific surface-exposed peptide sequences, previously shown to improve the retinal transduction properties in the context of AAV2. To this end, HROs were transduced with increasing concentrations of rAAV9, rAAV9.GL, or rAAV9.NN carrying a self-complementary genome with a cytomegalovirus-enhanced green fluorescent protein (eGFP) cassette and were monitored for eGFP expression. The rAAV vectors transduced HROs in a dose-dependent manner, with rAAV9.NN achieving the highest efficiency and fastest onset kinetics, leading to detectable eGFP signals in photoreceptors, some interneurons, and Müller glia already at 2 days post-transduction. The potency-enhancing effect of the NN peptide insert was replicated when using the corresponding AAV2-based version (rAAV2.NN). Taken together, we report the application of an HRO system for screening novel AAV vectors and introduce novel vector candidates with enhanced transduction efficiency for human retinal cells.

Reporter gene knock-in into Marc-145 cells using CRISPR/Cas9-mediated homologous recombination.

Marc-145 cells (monkey embryonic kidney epithelial cells) play a critical role in the biotechnology industry as certain virus host cells. To investigate the expression of enhanced green fluorescent protein (eGFP) gene as a foreign gene in Marc-145 cells, which we developed an approach of foreign gene site-specific knock-in into Marc-145 cells by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and putatively explored appropriate genomic recombination sites in Marc-145 cells. Our study demonstrated that the specific homologous recombination (HR) site between the Rac GTPase activating protein 1 (RACGAP1) and the acid-sensing ion channel subunit 1 (ASIC1) genes of the 11th chromosome could be used as the target site of Cas9 for the generation of target gene knock-in into Marc-145 cells, by the insertion of the eGFP cassette into the specific HR site and subsequent expression. Junction PCR, sequencing, Southern blot and fluorescence assay determined eGFP gene-specific knock-in HR site between the RACGAP1 and ASIC1 genes of the 11th chromosome, which was identified by the genomic safe harbours in Marc-145 cells. Our study encouraged a broader range of applications, such as Marc-145 cells development and engineering for virus adaption and yield increase in the vaccine biotechnology industry.

Foamy Virus Based Vectors for CAR-T Cell Development

Introduction A novel approach that can cover the therapeutic gap in NHL treatment are the autologous T cells, expressing Chimeric Antigen Receptors (CAR-T cells) against tumor markers. Such clinical-grade products based on Lenti (LV) or Retro- vectors have hit the market. An alternative vector system for CAR gene transfer in T-cells are Foamy Viruses (FV). To evaluate the potential of FV vectors in CAR-T cell development, we synthesized an antiCD19 scFv cDNA and cloned it in both an FV and an LV backbone; both vectors were tested in paired experiments Material and Methods The anti-CD19 CAR was under the control of the EF1a promoter; EGFP expression was under the control of an IRES2 element. The anti-CD19 CAR sequence was deduced from published data. FV vectors were made with a 4-plasmid vector system in 293T cells. 2nd generation LV vectors were purchased from Addgene. Cord blood (CB), healthy donor peripheral blood (PB) and CLL patients' PB was used as a source for CD3+ cells using immunomagnetic enrichment. Informed consent has been obtained in all cases of human sample use. T cells were activated by antiCD3/CD28 beads and transduced with antiCD19 LV or FV vectors. Transduction efficiency was assayed by flow cytometry (FCM) using a PE-conjugated anti-mouse Fab antibody. FV and LV CAR-T cells were expanded with Rapid Expansion Protocol (REP) and their cytotoxicity assays was evaluated against the CD19+ cell lines Raji and Daudi. The CLL patient derived CAR-Ts were evaluated against autologous B cells. Cytotoxicity was evaluated with an FCM protocol using CFSE-stained target cells vs unstained effector CARTs in different ratios. At the end of the incubation cells were stained with 7AAD to discriminate against live/dead cells. CAR-T cell activation was also assayed by INF-γ ELISA, following cocultures with target cells at a ratio of 1:1 for 24h. Results Vector titers: LV vector titers were between 3-5x10^5 TU/ml for both LV vectors (with or without EGFP cassette). FV vector titers were between 2-4x10^5 TU/ml regardless of the presence of the EGFP cassette. Tx efficiency: FV can mediate efficient gene transfer on T cells in the presence of heparin at an effective dose of 20-40 U/ml using a spinoculation technique. Transduction efficiency ranged from 40-65% at MOI=3-5, and was comparable to the transduction efficiency of LV vectors at a much higher MOI (10 to 30). Cytotoxicity data on lines: Following REP, the cell population consisted mostly (close to 96% purity) of CAR-T cells regardless of the vector used or of the T cell source. Effector cells were cocultured with the CD19+ cell lines, Daudi and Raji at varying ratios. With cord blood derived FV-CAR-T cells, at 4h post coculture we observed a 39.4% cell lysis at a ratio of 10:1 effector to target (n=1). Similar results were obtained for LV vectors. Peripheral blood derived CAR-T cells at THE same ratio (10:1), demonstrated 83.9% and 93.1% cell lysis for FV-CART and LV-CART cells respectively (n=2). Cytotoxicity data on CLL cells: T-cells from peripheral blood of CLL patients were used to generate LV- and FV-CAR-T cells. At the ratio of 10:1, we observed 73.1% and 69,8% cytotoxicity for FV-CAR-Ts and 70.1% and 70.7% with LV-CAR-Ts, in 2 independent paired experiments. IFN as activation marker: In two paired activation experiments, CB-derived FV-CAR-T cells secrete 560 and 437pg/ml of IFN-γ; similarly, LV-CAR-Ts secrete 534 and 554pg/ml IFN-γ. Untransduced control cells, produced 68pg/ml and 12pg/ml for FV-CAR-T and LV-CAR-T experimental arm respectively. Conclusion In the current work, we developed and tested FV vectors for anti- CD19 CAR-T cell production. We proved that FV viral vectors are capable of mediating efficient gene transfer to human T cells. We developed a method to efficiently transfer FV vectors into T-cells, using a clinically relevant protocol with heparin. The FV-derived CAR T cells demonstrate the same cytotoxic properties in vitro as their LV-derived counterpart and the same activation levels in the presence of CD19 expressing target cells as measured by IFN-γ secretion. FV CARTs derived from PB of CLL patients were capable of mediating comparable cytotoxicity levels as their LV-derived counterparts. Overall, we provide a proof of concept that FVs could be a safe and efficient alternative to LV derived vectors for CAR-T cells. Disclosures No relevant conflicts of interest to declare.

Expression and hydroxyurea-triggered induction of EGFP upon CRISPR/Cas9-mediated integration into the γ-globin gene of K562 cells.

To knock-in an EGFP cassette into the γ-globin genes of K562 cells via CRISPR/Cas9, and to assess expression and hydroxyurea (HU)-mediated induction of the targeted EGFP transgene. The EGFP cassettes were specifically knocked into the Gγ gene. EGFP expression was detected in the targeted cell population and isolated clones. Furthermore, EGFP transcript and fluorescence levels were significantly induced following HU-treatment. This system is readily utilizable for genome scale studies of cis-acting regulatory elements which are implicated in γ-globin expression or HU-mediated induction.

3170 - Variation in HoxA9 Expression in the NUP98-HOXD13 Myelodysplasia Model, Using a Novel HoxA9-eGFP Reporter Mouse

HoxA9 is normally expressed in haematopoietic stem and progenitor cells of humans and of mice, but expression ceases in differentiated cells. Many patients with a variety of myeloid and lymphoid malignancies overexpress HoxA9 in their disease cells, a fact reflected in many mouse models of these diseases. HoxA9 overexpression is thought to drive a differentiation block or abnormal self-renewal in these cells, promoting the oncogenic phenotype. We sought to produce a transgenic mouse that reports expression of the HoxA9 gene from the endogenous locus via co-expression of a fluorescent marker, in hopes that this might facilitate the better understanding of the origins of disease. We designed a targeting strategy using CRISPR-Cas9 technology, inserting an eGFP cassette immediately before the endogenous HoxA9 stop codon, separated from the HoxA9 coding sequence by a P2A self-cleaving sequence. We used this strategy to generate transgenic mice and found that the resultant mice contained the expected sequence and were developmentally normal and fertile. We characterised the expression of eGFP in the haematopoietic system of 6 week old mice using flow cytometry and found that, as expected, mature blood cells were eGFP negative while within the stem and progenitor compartments there were consistent, discrete HoxA9-positive and -negative compartments. We sorted these cells and confirmed by RQ-PCR analysis that eGFP expression did indeed correlate with expression of HoxA9. We subsequently crossed these mice with NUP98-HOXD13 transgenic mice, an established model of MDS characterised by HoxA9 overexpression in progenitor and mature cells, and found a markedly higher percentage of eGFP positive stem and progenitor cells, and that the positive cells were also markedly brighter. eGFP expression levels within the stem and progenitor compartments were more variable in the NHD13 mice, perhaps suggesting variable growth of a clonal population. As expected, we also observed expression of eGFP in certain mature cell compartments. These mice represent a valuable tool for studying the role of HoxA9 in haematopoiesis.

Endothelial Progenitor Cells Transplantation Promotes the Whole Brain Recovery after Focal Ischemic Stroke

Background: Ischemic stroke caused severe damages to patients, mostly treatments for ischemic stroke focused recovery on the peri-infarct areas. In this study, we found that focal ischemic stroke caused damages to whole brain, endothelial progenitor cells (EPCs) treatment could significantly promote the recovery of the whole brain after ischemic stroke. Methods: EPCs were infected with lentivirus carrying luc2 and eGFP cassette for bioluminescence imaging (BLI) and labeled with Alkyl-PEI-SPIO for magnetic resonance imaging (MRI). 5×10^5 EPCs were transplanted intracardially 24 hours after photothrombotic ischemic stroke onset. BLI, MRI and behavior test were carried out at different time points. Expression of BDNF, VEGF in the ipsilateral and contralateral hemisphere and infarct volume were analyzed 7 days after EPCs transplantation. Expression of myelin basic protein (MBP) and NeuN in the ipsilateral and contralateral hemisphere were detected 14 days after EPCs transplantation. Findings: EPCs transplantation significantly increased the expression of BDNF, MBP and NeuN in both ipsilateral and contralateral hemisphere, while no significant differences were found in VEGF expression. Also, in vivo MRI results showed that fiber counts and FA values in both ipsilateral and contralateral corpus callosum increased 14 days after EPCs transplantation. No significant differences were found in the infarct volume 7 days after EPCs transplantation. mNSS scores and foot-faults test indicated better functional recovery 14 days after EPCs transplantation. Interpretation: Studies should focus on the whole brain recovery after stroke onset. EPCs treatment offered a powerful therapy method for whole brain recovery after ischemic stroke which may associated with elevated BDNF expression. Funding Statement: This work was supported by the National Key Basic Research Program of China (NOs. 2013CB733800 and 2013CB733803), the National Natural Science Foundation of China (NOs. 81230034 and 81520108015), the National Natural Science Foundation of Jiangsu Province (NO. BK20130057), the National Science Foundation for Youths of China (NOs. 81601544 and 81501522), the Fundamental Research Funds for the Central Universities (NO. 2242018k30004). C. Wang acknowledged the financial support from the China Scholarship Council for his joint PhD scholarship (NO. 201706090204). The funder had no role in the study design, data analysis, write-up or in the writing of the report. The corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. Declaration of Interest: The authors have no conflicts of interest to disclose. Ethics Approval Statement: This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Southeast University (approval ID: SYXK-2010.4987). BALB/c nude female mice (8 weeks, 20-23 g, Comparative Medicine Centre of Yangzhou University [SCXK (SU) 2012-0004], Yangzhou, China) were used in this study. Briefly, EPCs were isolated from newborns with all mothers’ informed consent and with the approval of the local hospital ethics committee.

One-Step piggyBac Transposon-Based CRISPR/Cas9 Activation of Multiple Genes.

Neural cell fate is determined by a tightly controlled transcription regulatory network during development. The ability to manipulate the expression of multiple transcription factors simultaneously is required to delineate the complex picture of neural cell development. Because of the limited carrying capacity of the commonly used viral vectors, such as lentiviral or retroviral vectors, it is often challenging to perform perturbation experiments on multiple transcription factors. Here we have developed a piggyBac (PB) transposon-based CRISPR activation (CRISPRa) all-in-one system, which allows for simultaneous and stable endogenous transactivation of multiple transcription factors and long non-coding RNAs. As a proof of principle, we showed that the PB-CRISPRa system could accelerate the differentiation of human induced pluripotent stem cells into neurons and astrocytes by triggering endogenous expression of different sets of transcription factors. The PB-CRISPRa system has the potential to become a convenient and robust tool in neuroscience, which can meet the needs of a variety of in vitro and in vivo gain-of-function applications.

Development of a fluorescent ASFV strain that retains the ability to cause disease in swine

African swine fever is a contagious and often lethal disease for domestic pigs with a significant economic impact for the swine industry. The etiological agent, African swine fever virus (ASFV), is a highly structurally complex double stranded DNA virus. No effective vaccines or antiviral treatment are currently commercially available. We present here the development of a strain of ASFV that has been shown to retain its ability to cause disease in swine, efficiently replicate in swine macrophage and that is fluorescently tagged. The insertion of an EGFP cassette replacing the reading frames for two neighboring genes, MGF360-13L and MGF360-14L, in highly virulent field isolate Georgia/2007, did not affect virus replication in cell cultures and did not affect disease progression in swine, the natural host for ASFV. A virulent fluorescently tagged ASFV is a suitable tool to conduct pathogenesis studies in swine, study on virus-macrophage interaction and to run large scale screens that require a sensitive high throughput output. Utilizing an EGFP reporter system for observing ASFV replication and infectivity can circumvent the time and labor-intensive steps associated with viral antigen-based assays such as the observation of hemadsorption or cytopathic effect.

Abstract 15113: A Novel Purification Method for Cardiomyocytes and Endothelial Cells Derived From Human Pluripotent Stem Cells Using Microrna-responsive Messenger Rna

MicroRNAs (miRNAs) are small non-coding RNA that controls gene expression through translational regulation or mRNA cleavage. Recent progress in synthetic biology demonstrated that synthetic “RNA switches” enable to regulate gene expression depending on the intracellular environment. Here we show that synthetic mRNA technologies that modulate reporter protein expression by sensing miRNA activities enable purification of target cells in a safe and effective manner. To select cardiac-specific miRNA candidates, we performed miRNA microarray analyses using human induced pluripotent stem cell (hiPSC) line integrated MYH6 promoter driven EGFP cassette. We found 14 candidates, and synthesized individually these 14 miRNA-responsive OFF switch mRNAs (miR-switches) containing the sequence of blue fluorescent protein (TagBFP). We individually transfected them with EGFP mRNA into differentiated cells including cardiomyocytes derived from human pluripotent stem cell (hPSCs). We found that several cells transfected with miR-1- or miR-208-switches diminished TagBFP intensity, and sorting of these cells yielded high populations of troponin T-positive cardiomyocytes reproducibly and significantly (e.g., up to >98% in five hPSC lines). In addition, we developed a non-sorting system for purification of cardiomyocytes using miR-switch that code apoptosis-inducible protein (up to >97%). These cardiomyocytes purified by miR-switches were successfully engrafted in mouse heart and did not form tumor even when injected into testes of SCID mice. We next examined whether the system can be applied to purify endothelial cells, and confirmed that several miR-switches enabled high purification of CD31+ cells from differentiated derivatives of hPSCs (up to >98%), which have the function of tube formation like HUVEC. Moreover, the use of multiple miRNA-responsive mRNAs enabled simultaneous and high purification of cardiomyocytes and endothelial cells from heterogeneous population derived from hPSCs induced by single differentiation method (up to >98%). Our results suggest that miR-switch technology can be used for a variety of purposes including purification in any type of cells.

Single-Cell Analysis of Proxy Reporter Allele-Marked Epithelial Cells Establishes Intestinal Stem Cell Hierarchy

SummaryThe recent development of targeted murine reporter alleles as proxies for intestinal stem cell activity has led to significant advances in our understanding of somatic stem cell hierarchies and dynamics. Analysis of these reporters has led to a model in which an indispensable reserve stem cell at the top of the hierarchy (marked by Bmi1 and Hopx reporters) gives rise to active intestinal stem cells (marked by an Lgr5 reporter). Despite these advances, controversy exists regarding the specificity and fidelity with which these alleles distinguish intestinal stem cell populations. Here, we undertake a comprehensive comparison of widely used proxy reporters including both CreERT2 and EGFP cassettes targeted to the Lgr5, Bmi1, and Hopx loci. Single-cell transcriptional profiling of these populations and their progeny reveals that reserve and active intestinal stem cells are molecularly and functionally distinct, supporting a two-stem-cell model for intestinal self-renewal.

Efficient disruption of Zebrafish genes using a Gal4-containing gene trap

BackgroundExternal development and optical transparency of embryos make zebrafish exceptionally suitable for in vivo insertional mutagenesis using fluorescent proteins to visualize expression patterns of mutated genes. Recently developed Gene Breaking Transposon (GBT) vectors greatly improve the fidelity and mutagenicity of transposon-based gene trap vectors.ResultsWe constructed and tested a bipartite GBT vector with Gal4-VP16 as the primary gene trap reporter. Our vector also contains a UAS:eGFP cassette for direct detection of gene trap events by fluorescence. To confirm gene trap events, we generated a UAS:mRFP tester line. We screened 270 potential founders and established 41 gene trap lines. Three of our gene trap alleles display homozygous lethal phenotypes ranging from embryonic to late larval: nsf tpl6, atp1a3atpl10 and flrtpl19. Our gene trap cassette is flanked by direct loxP sites, which enabled us to successfully revert nsf tpl6, atp1a3atpl10 and flrtpl19 gene trap alleles by injection of Cre mRNA. The UAS:eGFP cassette is flanked by direct FRT sites. It can be readily removed by injection of Flp mRNA for use of our gene trap alleles with other tissue-specific GFP-marked lines. The Gal4-VP16 component of our vector provides two important advantages over other GBT vectors. The first is increased sensitivity, which enabled us to detect previously unnoticed expression of nsf in the pancreas. The second advantage is that all our gene trap lines, including integrations into non-essential genes, can be used as highly specific Gal4 drivers for expression of other transgenes under the control of Gal4 UAS.ConclusionsThe Gal4-containing bipartite Gene Breaking Transposon vector presented here retains high specificity for integrations into genes, high mutagenicity and revertibility by Cre. These features, together with utility as highly specific Gal4 drivers, make gene trap mutants presented here especially useful to the research community.

PiggyBac-mediated generation of stable transfectants with surface human leukocyte antigen expression from a small number of cells

Techniques useful for isolating a series of stable transfectants from a small number of cells are important for high-throughput functional analyses of genes of interest (GOIs). This study employed piggyBac transposon vectors carrying an IRES–eGFP cassette to isolate stable transfectants. The 14 transfections performed produced stable transfectants; a small number of cells (5.7×104) was sufficient even when liposomes were used to transfect non-adherent cells. Of note, there was a close relationship between eGFP fluorescence intensity and GOI (human leukocyte antigen) expression level. Therefore, the current system would be useful for the high-throughput acquisition of clones showing high GOI expression.

The epimastigote stage-specific gene expression of CESP is tightly regulated by its 3′ UTR

It is known that gene expression in kinetoplastids is regulated post-transcriptionally. Although previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3′ untranslated region (UTR) of mRNA and also by RNA binding proteins that use bloodstream and procyclic forms, no studies have been performed in the epimastigote form (EMF) of African trypanosomes. This study shows that the cis-elements of the congolense epimastigote-specific protein (cesp) gene regulate its EMF-specific expression. Four different egfp expression cassettes containing 5′ and 3′ UTRs derived from actin and cesp were integrated into the Trypanosoma congolense genome. EGFP expression was observed in EMF trypanosomes when the egfp cassette contained the cesp 3′ UTR. These results indicate that the essential cis-element located in the cesp 3′ UTR selectively stabilizes mRNA in the EMF stage and/or destabilized mRNA in other stages. This is the first report of post-transcriptional gene expression regulation in EMF African trypanosomes.

Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

Many models of human tauopathies have been generated in mice by expression of a human mutant tau with maintained expression of mouse endogenous tau. Because murine tau might interfere with the toxic effects of human mutant tau, we generated a model in which a pathogenic human tau protein is expressed in the absence of wild-type tau protein, with the aim of facilitating the study of the pathogenic role of the mutant tau and to reproduce more faithfully a human tauopathy. The Tg30 line is a tau transgenic mouse model overexpressing human 1N4R double-mutant tau (P301S and G272V) that develops Alzheimer's disease-like neurofibrillary tangles in an age-dependent manner. By crossing Tg30 mice with mice invalidated for their endogenous tau gene, we obtained Tg30xtau(-/-) mice that express only exogenous human double-mutant 1N4R tau. Although Tg30xtau(-/-) mice express less tau protein compared with Tg30, they exhibit signs of decreased survival, increased proportion of sarkosyl-insoluble tau in the brain and in the spinal cord, increased number of Gallyas-positive neurofibrillary tangles in the hippocampus, increased number of inclusions in the spinal cord, and a more severe motor phenotype. Deletion of murine tau accelerated tau aggregation during aging of this mutant tau transgenic model, suggesting that murine tau could interfere with the development of tau pathology in transgenic models of human tauopathies.