Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function. However, contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported. Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system. We demonstrated the feasibility of this strategy at sox10 and isl1 loci, and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter, allowing generation of genetic mosaics for lineage tracing. We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles, both tagged with two different fluorescent reporters. By introducing Cre recombinase, these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel; furthermore, differential fluorescent labeling of the positive and negative alleles enables simple, early and efficient real-time discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes. We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus. Furthermore, we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology. Our system could easily be expanded for other applications or to other organisms, and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.
Alleles ; Animals ; CRISPR-Cas Systems ; DNA End-Joining Repair ; DNA, Circular/metabolism* ; Embryo, Nonmammalian ; Gene Editing/methods* ; Gene Knock-In Techniques ; Gene Knockout Techniques ; Genes, Reporter ; Genetic Loci ; Genotyping Techniques ; Green Fluorescent Proteins/metabolism* ; Integrases/metabolism* ; Luminescent Proteins/metabolism* ; Mutagenesis, Insertional ; Single-Cell Analysis ; Zebrafish/metabolism*

CRISPR-Cas Systems ; Cell Survival ; Genetic Loci ; genetics ; Genome ; genetics ; HEK293 Cells ; Humans ; Luminescent Proteins ; analysis ; Optical Imaging ; methods

OBJECTIVETo label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits.

METHODSGFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen.

RESULTSThe expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen.

CONCLUSIONSThe hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

Animals ; Cartilage, Articular ; metabolism ; Cells, Cultured ; Chondrocytes ; metabolism ; Flow Cytometry ; Genetic Markers ; Genetic Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Luminescent Agents ; RNA, Messenger ; analysis ; Rabbits

To study the effects and possible mechanism of Vitamin K(2) (VK(2)) in the treatment of MDS-JSN04 cells, the changes of morphologic features of MDS-JSN04 cells were investigated by cytomorphology, the apoptosis of MDS-JSN04 cells was observed by transmission electron microscope; cellular proliferation was determined by the MTT assay; cell apoptosis, cell cycle shift and expression of myeloid-specific differentiation antigen (CD11b, CD13) were analyzed by flow cytometry (FCM). The expression of apoptosis-related genes bcl-2, survivin and bax were detected by retrotranscriptase polymerase chain reaction (RT-PCR); the activity of caspase-3 was determined by chemiluminescence assay. The results showed that the typical apoptotic morphological features appeared in cells treated with VK(2) for 72 hours; VK(2) induced apoptosis of MDS-JSN04 cells and in a dose-and-time-dependent manner, G(0)/G(1) cell arrest and significantly down-regulated the expression of bcl-2 and survivin, but had no effect on the expression of bax; the activity of caspase-3 significantly increased. It is concluded that VK(2) induces apoptosis of MDS-JSN04 cells through activating caspase-3 pathways and the apoptosis-related genes bcl-2, survivin may play an important role in this process.
Apoptosis ; drug effects ; CD11b Antigen ; analysis ; CD13 Antigens ; analysis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; Inhibitor of Apoptosis Proteins ; Luminescent Measurements ; methods ; Microscopy, Electron, Transmission ; Microtubule-Associated Proteins ; genetics ; Myelodysplastic Syndromes ; genetics ; metabolism ; pathology ; Neoplasm Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Vitamin K 2 ; pharmacology ; bcl-2-Associated X Protein ; genetics

OBJECTIVETo develop a system for quick screening of efficient siRNA targeted HBx mRNA.

METHODSUsing recombination DNA technique, the fusion expression plasmid of HBx and EGFP was constructed, and siRNA expression cassettes (SECs) containing U6+1, H1 or tRNA(Val )promoter were prepared via one-step overlapping extension PCR. By co-transfection with recombinant plasmid and SECs into AD293 cell, the inhibition effects on the transient expression of HBx-EGFP fusion protein were analyzed by FACS and semi-quantitated RT-PCR analysis.

RESULT(1)HBx-EGFP fusion protein expression plasmid pHBx-EGFP was constructed successfully, which expressed green fluorescence in cell mainly located at plasma or the periphery of nucleus in granules. (2) Co-transfection with recombinant plasmid and SECs into AD293 cells resulted in inhibition of HBx-EGFP expression. SEC-siHBx388 showed significant inhibition effect on HBx-EGFP expression compared with SEC-siHBx271, indicating that siHBx388 is effective siRNA site and could be screened out with our screening system. In addition,the results of that U6+1-, tRNA(Val) and H1-siHBx388 reduced HBx-EGFP expression by 21.7%, 12.9% and 12.4% of control respectively indicated that both tRNAVal and H1 promoter was high efficient in driving effect of siHBx388.

CONCLUSIONCombination of the HBx expression carrying reporter gene and PCR-based multi promoter SECs may develop a useful system to be applied in identification of optimal HBx- siRNA and its matching promoter.

Base Sequence ; Cells, Cultured ; Genetic Therapy ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; genetics ; Molecular Sequence Data ; Plasmids ; RNA, Small Interfering ; analysis ; Recombinant Fusion Proteins ; biosynthesis ; Trans-Activators ; antagonists & inhibitors ; genetics ; Transfection

Visualizing living cells growing on co-cultured biomaterials is ideal for material biocompatibility evaluation in vitro. In this experiment, mouse fibroblasts L929 were labeled by introducing the gene coding enhanced green fluorescent protein (EGFP) marker into the cells. Morphology as well as proliferation of labeled cells surrounding or on the surface of co-cultured denture base resin slides were observed by use of phase-contrast microscope and fluorescent microscope directly. It was found that residual methyl methacrylate (MMA) in the denture base resin exhibited transient cytotoxicity to fibroblasts and this transient cytotoxicity could be eliminated by pre-extracting the resin with ddH2O for a short time. This fact demonstrated that even slight cytotoxicity of materials could be detected through imaging of living cells near material or material touched. And it was suggested that imaging of living cells co-cultured with biomaterial is helpful to understanding biocompatibility of materials more accurately.
Acrylic Resins ; Animals ; Biocompatible Materials ; Cell Division ; Cells, Cultured ; Culture Media ; Dental Materials ; Denture Bases ; Fibroblasts ; cytology ; Green Fluorescent Proteins ; Indicators and Reagents ; analysis ; Luminescent Proteins ; genetics ; Mice ; Polymethyl Methacrylate ; Transfection

To investigate the transduction efficiency of recombinant adeno-associated virus 2 (rAAV-2) in human umbilical cord blood CD34(+) hematopoietic stem/progenitor cells, the CD34(+) cells sorted by the method of magnetic cell sorting from human cord blood were infected with the rAAV-2 expressing the green fluorescent protein (GFP) gene. After transduction for 19 hours, the expression of GFP was detected under fluorescence microscope. The results showed that 43% CD34(+) cells expressed the GFP gene at a multiplicity of infection of 2 x 10(5). It is concluded that the rAAV-2 can transduce human cord blood CD34(+) hematopoietic stem/progenitor cells efficiently.
Antigens, CD34 ; analysis ; Dependovirus ; genetics ; Fetal Blood ; cytology ; Genetic Therapy ; Green Fluorescent Proteins ; Hematopoietic Stem Cells ; metabolism ; Humans ; Luminescent Proteins ; genetics ; Recombination, Genetic ; Transduction, Genetic

OBJECTIVETo examine the potential function of NMDA receptor NR2A subunit C-terminus in assembling and surface expression of the receptor in HEK293 cells.

METHODSFive vectors GFP- NR2ADeltaC1- DeltaC5 were constructed for expressing N-terminally GFP-tagged NR2A with C-terminal deletion at different regions by using conventional techniques of molecular cloning. The deleted region for NR2ADeltaC1-Delta C5 was 897L-1017S, 1024D-1142P, 1149D-1347G, 1354S-1464V, and 897L-1464V. These plasmids were transfected alone or co-transfected with NR1-1a into HEK293 cells. The surface NMDA receptors were immuno-stained using rabbit antibody against GFP and Cy3 conjugated secondary antibody in living cells.

RESULTThe vectors GFP-NR2ADeltaC1-DeltaC5 were generated and all of them expressed GFP fluorescence in the transfected cells. Surface NMDA receptors were detected by immuno-labeling with anti-GFP in the cells co-transfected by NR1-1a and any one of GFP-NR2ADeltaC1-DeltaC5. However, no surface expression of NR2A proteins was found in the transfected cells with any one of these plasmids alone.

CONCLUSIONWithin the region downstream from the 897L of NR2A subunit, neither a particular domain directly interacted with ER retention domain in NR1-1a C1 cassette, nor that determining ER retention of NR2A subunit itself has been found, indicating that more complicated mechanisms might exist in which the subunit assembling and targeting to plasma membrane of NMDA receptors undergo.

Cell Line ; Gene Deletion ; Green Fluorescent Proteins ; Humans ; Luminescent Proteins ; metabolism ; Mutation ; Receptors, N-Methyl-D-Aspartate ; analysis ; genetics

To determine the feasibility of a nonradioactive electrophoresis mobility shift assay for detecting nuclear transcription factor, double-stranded oligonucleotides encoding the consensus target sequence of NF-kappa B were labelled with DIG by terminal transferase. After nuclear protein stimulated with phorbol 12-myristate 13-acetate (PMA) or PMA and pyrrolidine dithiocarbamate (PDTC) electrophoresed on 8% nondenaturing poliacrylamide gel together with oligeonucleotide probe, they were electro-blotted nylon membrane positively charged. Anti-DIG-AP antibody catalyzed chemiluminescent substrate CSPD to image on X-film. The results showed that nuclear proteins binded specifically to the NF-kappa B consensus sequence in the EMSA by chemiluminescent technique method and the activity of NF-kappa B in PMA group was more than that in PMA + PDTC group. It is suggested that detection of NF-kappa B by EMSA with chemiluminescent technique is feasible and simple, which can be performed in ordinary laboratories.
Animals ; DNA-Binding Proteins ; analysis ; genetics ; Electrophoretic Mobility Shift Assay ; Luminescent Measurements ; NF-kappa B ; analysis ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Trans-Activators ; analysis ; genetics ; Transcription, Genetic ; Transcriptional Activation

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.
Adenoviruses, Human/isolation & purification* ; Animals ; Blood-Brain Barrier ; Brain/virology* ; Cerebellum/cytology ; Cerebellum/virology ; Comparative Study ; Female ; Genes, Reporter ; Genetic Vectors/administration & dosage ; Genetic Vectors/isolation & purification ; Genetic Vectors/pharmacokinetics* ; Hippocampus/virology ; Inferior Colliculus/virology ; Injections, Intravenous ; Luminescent Proteins/analysis ; Luminescent Proteins/biosynthesis ; Luminescent Proteins/genetics ; Mice ; Mice, Inbred BALB C ; Neuroglia/virology ; Neurons/virology* ; Purkinje Cells/virology ; Pyramidal Cells/virology ; Recombinant Fusion Proteins/analysis ; Recombinant Fusion Proteins/biosynthesis ; Recombinant Fusion Proteins/genetics ; Tail/blood supply ; Tissue Distribution