Red Fluorescent Protein Gene Research Articles

Targeted Expression in Zebrafish Primordial Germ Cells by Cre/loxP and Gal4/UAS Systems

In zebrafish and other vertebrates, primordial germ cells (PGCs) are a population of embryonic cells that give rise to sperm and eggs in adults. Any type of genetically manipulated lines have to be originated from the germ cells of the manipulated founders, thus it is of great importance to establish an effective technology for highly specific PGC-targeted gene manipulation in vertebrates. In the present study, we used the Cre/loxP recombinase system and Gal4/UAS transcription system for induction and regulation of mRFP (monomer red fluorescent protein) gene expression to achieve highly efficient PGC-targeted gene expression in zebrafish. First, we established two transgenic activator lines, Tg(kop:cre) and Tg(kop:KalTA4), to express the Cre recombinases and the Gal4 activator proteins in PGCs. Second, we generated two transgenic effector lines, Tg(kop:loxP-SV40-loxP-mRFP) and Tg(UAS:mRFP), which intrinsically showed transcriptional silence of mRFP. When Tg(kop:cre) females were crossed with Tg(kop:loxP-SV40-loxP-mRFP) males, the loxP flanked SV40 transcriptional stop sequence was 100 % removed from the germ cells of the transgenic hybrids. This led to massive production of PGC-specific mRFP transgenic line, Tg(kop:loxP-mRFP), from an mRFP silent transgenic line, Tg(kop:loxP-SV40-loxP-mRFP). When Tg(kop:KalTA4) females were crossed with Tg(UAS:mRFP) males, the hybrid embryos showed PGC specifically expressed mRFP from shield stage till 25 days post-fertilization (pf), indicating the high sensitivity, high efficiency, and long-lasting effect of the Gal4/UAS system. Real-time PCR analysis showed that the transcriptional amplification efficiency of the Gal4/UAS system in PGCs can be about 300 times higher than in 1-day-pf embryos. More importantly, when the UAS:mRFP-nos1 construct was directly injected into the Tg(kop:KalTA4) embryos, it was possible to specifically label the PGCs with high sensitivity, efficiency, and persistence. Therefore, we have established two targeted gene expression platforms in zebrafish PGCs, which allows us to further manipulate the PGCs of zebrafish at different levels.

Optimal place of a foreign gene in the genome of viral haemorrhagic septicaemia virus (VHSV) for development of VHSV-based viral-vectored vaccines

As the strength and duration of immune responses can be regulated by the antigen dose, higher expression of foreign antigens in the viral-vectored vaccines would be an important factor for inducing effective immune responses. The aim of this study was to determine the optimal insertion place of a foreign antigen gene in the genome of viral haemorrhagic septicaemia virus (VHSV) for development of VHSV-based viral-vectored vaccines. Recombinant VHSVs (rVHSVs) harbouring the red fluorescent protein (RFP) gene between N and P (rVHSV-A-RFP), P and M (rVHSV-B-RFP), or M and G genes (rVHSV-C-RFP) in the genome were rescued by reverse genetics. Their replication ability and expression level of RFP were compared according to the inserted locations. The viral titres of each rVHSV were not significantly different. However, Epithelioma papulosum cyprini (EPC) cells infected with rVHSV-A-RFP or rVHSV-B-RFP showed clearly higher fluorescence than cells infected with rVHSV-C-RFP. There was no significant difference in RFP expression between cells infected with rVHSV-A-RFP and rVHSV-B-RFP. The present results indicate that insertion of a foreign gene between N and P, or P and M genes of VHSV genome would be advantageous for development of VHSV-based viral-vectored vaccines. The present work is the first report on the optimal location of a foreign gene in VHSV genome for high expression, and the locations identified in this study would be suitable for the development of VHSV-based viral-vectored vaccines.

Effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 gene and bone morphogenetic protein-7 gene on osteogenic differentiation of rat adipose-derived mesenchymal stromal cells

Objective To investigate the effects of adenovirus-mediated co-transfection of bone morphogenetic protein-2 (BMP-2) gene and bone morphogenetic protein-7 (BMP-7) gene on osteogenic differentiation of adipose-derived mesenchymal stromal cells (ADSCs).Methods The expression adenovirus vectors with green fluorescent protein gene carrying BMP-2 and adenovirus vectors with red fluorescent protein gene carrying BMP-7 were packaged and produced respectively.Rat adipose-derived stromal cells were isolated and cultured in vitro,and were transfected with Ad-BMP-2 (Ad-BMp-2 group),Ad-BMP-7 (Ad-BMP-7 group),co-transfected with Ad-BMP-2 and Ad-BMP-7 (Ad-BMP-2 ± Ad-BMP-7 group),or transfected with no virus (control group).The expression of osteocalcin (OCN) and collagen Ⅰ protein at 14th day after transfection was detected by using Western blotting in each group.Alkaline phosphatase (ALP) activity in ADSCs was measured.Results The expression of OCN and collagen Ⅰ protein in cotransfected group was significantly higher than that in the other groups (P ＜ 0.05).The ALP activity in cotransfected group was significantly stronger than that in the other groups (P ＜ 0.01).Conclusion Adenovirus-mediated co-transfection of BMP-2 gene and BMP-7 gene may promote osteogenic differentiation of adipose-derived ADSCs. Key words: Bone morphogenetic protein; Adenovirus; Adipose-derived stromal cells; Osteogenic differentiation

The effect of glioma stem/progenitor cell on tumor angiogenesis traced by red/green fluorescent protein

Objective To explore the potential role of glioma stem cell in the generation and maintenance of tumor blood vessels.Methods Gliomas stem cells (CD133 + tumor cells) transfected with red fluorescent protein (RFP) gene were implanted in situ stereotaxicly (25 μl,1 × 106 cells) or subcutaneously (200 μ1,1 × 106 cells) in nude mice,then the xenografts (2-3 weeks)were pressed fiat and tumor vessels structures were observed under fluorescence microscope and laser confocal microscope.Results Tissue squash method can clearly display the host normal vessels and some cells source of vascular endothelial cells inside the tumor region on the basis of different colors under fluorescence、laser confocal microscope.Images displayed vascular cells can be classified colors into three types:host derived cells(green),tumor derived(red),tumor derived and host-derived fusion(yellow).Cells form of tumor angiogenesis can be divided into three types:proliferation of endothelial cells(EC),tumor stem cells (TC),tumor stem cells and host-derived endothelial cells fusion (EC/TC).Conclusion Gliomas stem cells transfected with red fluorescent protein (RFP) gene were implanted in green fluorescent protein(GFP) transgenic nude mice model was very useful to study the processes of malignant glioma angiogenesis.This model can prove the glioma stem/progenitor cells act as initiating cell in tumor angiogenesis. Key words: Tumor stem cells; Fluorescent protein gene transfection; Transgenic fluorescence nude mice; Transplantable tumor; Tumor angiogenesis

An alternative in vitro drug screening test using Leishmania amazonensis transfected with red fluorescent protein

Fluorescent and colorimetric reporter genes are valuable tools for drug screening models, since microscopy is labor intensive and subject to observer variation. In this work, we propose a fluorimetric method for drug screening using red fluorescent parasites. Fluorescent Leishmania amazonensis were developed after transfection with integration plasmids containing either red (RFP) or green fluorescent protein (GFP) genes. After transfection, wild-type (LaWT) and transfected (LaGFP and LaRFP) parasites were subjected to flow cytometry, macrophage infection, and tests of susceptibility to current antileishmanial agents and propranolol derivatives previously shown to be active against Trypanosoma cruzi. Flow cytometry analysis discriminated LaWT from LaRFP and LaGFP parasites, without affecting cell size or granulosity. With microscopy, transfection with antibiotic resistant genes was not shown to affect macrophage infectivity and susceptibility to amphotericin B and propranolol derivatives. Retention of fluorescence remained in the intracellular amastigotes in both LaGFP and LaRFP transfectants. However, detection of intracellular RFP parasites was only achieved in the fluorimeter. Murine BALB/c macrophages were infected with LaRFP parasites, exposed to standard (meglumine antimoniate, amphotericin B, Miltefosine, and allopurinol) and tested molecules. Although it was possible to determine IC50 values for 4 propranolol derivatives (1, 2b, 3, and 4b), all compounds were considered inactive. This study is the first to develop a fluorimetric drug screening test for L. amazonensis RFP. The fluorimetric test was comparable to microscopy with the advantage of being faster and not requiring manual counting.

The Potential of a Fluorescent-Based Approach for Bioassay of Antifungal Agents against Chili Anthracnose Disease in Thailand

Severe chili anthracnose disease in Thailand is caused by Colletotrichum gloeosporioides and C. capsici. To discover anti-anthracnose substances we developed an efficient dual-fluorescent labeling bioassay based on a microdilution approach. Indicator strains used in the assay were constructed by integrating synthetic green fluorescent protein (sGFP) and Discosoma sp. red fluorescent protein (DsRedExp) genes into the genomes of C. gloeosporioides or C. capsici respectively. Survival of co-spore cultures in the presence of inhibitors was determined by the expression levels of these fluorescent proteins. This developed assay has high potential for utilization in the investigation of selective inhibition activity to either one of the pathogens as well as the broad-range inhibitory effect against both pathogens. The value of using the dual-fluorescent assay is rapid, reliable, and consistent identification of anti-anthracnose agents. Most of all, the assay enables the identification of specific inhibitors under the co-cultivation condition.

Molecular Characterization of Cytoskeletal Beta-Actin and its Promoter in the Javanese Ricefish Oryzias javanicus

We characterized the cytoskeletal beta-actin (<TEX>${\beta}$</TEX>-ACT) gene (actb) and its 5'-upstream regulatory region in the Javanese ricefish Oryzias javanicus. The gene and protein structures were deduced from amino acid sequences of the actb gene and conserved in the teleost lineage. The O. javanicus actb gene has common transcription factor binding motifs in its regulatory region found in teleostean orthologues. Following quantitative reverse transcription-PCR, actb gene transcripts were detected in all tissues examined; however, the basal expression levels were different. During early development, O. javanicus actb mRNA levels showed a gradual increase and peaked between late somitogenesis and the heartbeat stage. Microinjection of O. javanicus embryos with the actb gene promoter-driven red fluorescent protein (RFP) gene reporter vector showed a ubiquitous distribution of RFP signals, although most exhibited a mosaic pattern of transgene expression. A small number of microinjected embryos displayed a wide distribution of RFP signals over their entire body, which resembled the expression pattern of endogenous actb. Data from this study provide a basis to develop a transgenic system with ubiquitous expression of foreign genes in O. javanicus.

Characterization of stable fluorescent transgenic marine medaka (Oryzias dancena) lines carrying red fluorescent protein gene driven by myosin light chain 2 promoter

Stable transgenic germlines carrying the red fluorescence protein (RFP) gene (rfp) driven by fast skeletal myosin light chain-2 gene (mlc2f) promoter were established in a truly euryhaline fish species, the marine medaka (Oryzias dancena; Beloniformes). Transgenic lines contained transgene copy numbers varying from a single copy to more than 230 copies per genome. Although the transgenic founders displayed mosaic and/or ectopic expression of the RFP signal, the resultant F1 transgenics and their progeny showed consistently stable transmission of the transgenic locus and uniform RFP signal through several subsequent generations. In adult transgenics, an authentic brilliant red fluorescence was achieved over the skeletal muscles of the transgenic individuals, which might be sufficient for ornamental display. Expression analysis of the transgenic mRNAs indicated that rfp transcripts were predominantly expressed in the skeletal muscles. Different transgenic lines displayed different levels of transgene expression at the mRNA, protein, and phenotypic levels. However, the efficiency of transgene expression was independent of the transgene copy number. The RFP protein levels were consistently stable in the transgenic fish muscles through several generations, up to F5. The results of this study suggest that transgenic marine medaka that acquire strong fluorescent signals in their skeletal muscles can be developed as a promising, novel ornamental fish for display in both freshwater and seawater aquaria.

Remnant living cells that escape cell loss in late-stage tumors exhibit cancer stem cell-like characteristics

A balance between cell proliferation and cell loss is essential for tumor progression. Although up to 90% of cells are lost in late-stage carcinomas, the progression and characteristics of remnant living cells in tumor mass are unclear. Here we used molecular imaging to track the progression of living cells in a syngeneic tumor model, and ex vivo investigated the properties of this population at late-stage tumor. The piggyBac transposon system was used to stably introduce the dual reporter genes, including monomeric red fluorescent protein (mRFP) and herpes simplex virus type-1 thymidine kinase (HSV1-tk) genes for fluorescence-based and radionuclide-based imaging of tumor growth in small animals, respectively. Iodine-123-labeled 5-iodo-2′-fluoro-1-beta-𝒟-arabinofuranosyluracil was used as a radiotracer for HSV1-tk gene expression in tumors. The fluorescence- and radionuclide-based imaging using the single-photon emission computed tomography/computed tomography revealed that the number of living cells reached the maximum at 1 week after implantation of 4T1 tumors, and gradually decreased and clustered near the side of the body until 4 weeks accompanied by enlargement of tumor mass. The remnant living cells at late-stage tumor were isolated and investigated ex vivo. The results showed that these living cells could form mammospheres and express cancer stem cell (CSC)-related biomarkers, including octamer-binding transcription factor 4, SRY (sex-determining region Y)-box 2, and CD133 genes compared with those cultured in vitro. Furthermore, this HSV1-tk-expressing CSC-like population was sensitive to ganciclovir applied for the suicide therapy. Taken together, the current data suggested that cells escaping from cell loss in late-stage tumors exhibit CSC-like characteristics, and HSV1-tk may be considered a theranostic agent for targeting this population in vivo.

Characterization of estrogen-responsive transgenic marine medaka Oryzias dancena germlines harboring red fluorescent protein gene under the control by endogenous choriogenin H promoter

Transgenic marine medaka (Oryzias dancena) germlines were generated by the microinjection of the red fluorescent protein (RFP) reporter gene (rfp) driven by the endogenous choriogenin H gene (chgH) promoter. The selected transgenic lines contained multiple copies of the transgene (3-42 copies per cell) in their genomes. Although all the founders were mosaic, the transgene was stably transmitted from the F1 generation to all subsequent generations following a Mendelian pattern. Different transgenic lines showed different responsiveness to estradiol-17β (E2) exposure at the mRNA and protein levels, and the expression efficiency was dependent upon the transgene copy number. The induction of RFP was significantly affected by the developmental stage of transgenic larvae: later-stage larvae (older than 7 days post-hatching) showed higher sensitivity to E2 exposure than earlier-stage larvae. The response of transgenic expression to E2 was fairly dependent upon the E2 dose (200-3,200 ng/L) and exposure period (1-7 days), according to both a microscopic examination of RFP intensity and a qRT-PCR assay. The transgenic marine medaka showed similar transgenic responses to E2 under freshwater, brackish, and seawater conditions. In addition to E2, the transgenic RFP signal was also successfully induced during 1-week exposure to various other natural (1 μg/L estrone and 10 μg/L estriol) and synthetic (xeno)estrogens (0.1 μg/L 17α-ethynylestradiol, 1 μg/L diethylstilbestrol, and 10 mg/L bisphenol A). The efficiency of transgene expression varied greatly among the chemicals tested. The results of this study suggest that the chgH-rfp transgenic marine medaka species will be useful in the in vivo detection of waterborne estrogens under a wide range of salinity conditions.

Genome Editing of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells With Zinc Finger Nucleases for Cellular Imaging

Molecular imaging has proven to be a vital tool in the characterization of stem cell behavior in vivo. However, the integration of reporter genes has typically relied on random integration, a method that is associated with unwanted insertional mutagenesis and positional effects on transgene expression. To address this barrier, we used genome editing with zinc finger nuclease (ZFN) technology to integrate reporter genes into a safe harbor gene locus (PPP1R12C, also known as AAVS1) in the genome of human embryonic stem cells and human induced pluripotent stem cells for molecular imaging. We used ZFN technology to integrate a construct containing monomeric red fluorescent protein, firefly luciferase, and herpes simplex virus thymidine kinase reporter genes driven by a constitutive ubiquitin promoter into a safe harbor locus for fluorescence imaging, bioluminescence imaging, and positron emission tomography imaging, respectively. High efficiency of ZFN-mediated targeted integration was achieved in both human embryonic stem cells and induced pluripotent stem cells. ZFN-edited cells maintained both pluripotency and long-term reporter gene expression. Functionally, we successfully tracked the survival of ZFN-edited human embryonic stem cells and their differentiated cardiomyocytes and endothelial cells in murine models, demonstrating the use of ZFN-edited cells for preclinical studies in regenerative medicine. Our study demonstrates a novel application of ZFN technology to the targeted genetic engineering of human pluripotent stem cells and their progeny for molecular imaging in vitro and in vivo.

Generation and characterization of a stable red fluorescent transgenic Tanichthys albonubes line

White cloud mountain minnow (Tanichthys albonubes) is a small cyprinid oviparous fresh-water fish in Southern China. For ornamental purpose, the red fluorescent protein (RFP) transgenic T. albonubes was generated by microinjection of a pMYLZ2-RFP gene construct containing zebrafish myosin light polypeptide 2 (mylz2) promoter into the fertilized eggs, and a transgenic line was established. For this line, segregation of RFP gene followed Mendelian single-gene inheritance, confirming transgene integration into the T. albonubes genome at a single locus. Results of reverse transcriptase-polymerase chain reaction (RT-PCR) analyses showed that RFP transgene was expressed in almost all body tissues, revealing an ectopic transgene expression. Based on ordinary and quantitative PCR analyses, integration pattern of RFP gene was determined to be three copies and head-to-tail multimers. An approximately 1.0 kb fragment amplified from 5'-flanking sequence of transgenes was identified in wildtype T. albonubes genome, and bears no homologous sequence in the GenBank database. In the 3'flanking region, an approximately 1.2 kb fragment with unidentified source was amplified which has 99% homology to enterobacteria phage P1. The above results well demonstrated the pMYLZ2-RFP constructs have being inserted as a stable DNA fragment in the T. albonubes genome and could be stably inherited and expressed in transgenic progenies.

Site-specific Bacterial Chromosome Engineering: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (Φ) C31. The ΦC31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively). This recombination is stable and does not revert. A "landing pad" (LP) sequence consisting of a spectinomycin-resistance gene, aadA (SpR), and the E. coli ß-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob. The expression of the ΦC31 integrase gene (cloned from pHS62) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK7813. A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

A dual color fluorescent reporter system for the real time detection of promoter activity

Understanding the mechanisms controlling transcription of a gene requires the identification and characterization of its cis-acting regulatory elements. A highly useful approach to the identification and characterization of cis-acting elements has been the systematic coupling of genomic fragments to reporter constructs, so called "promoter bashing". The expression from such reporters must be normalized for differences in transient transfection efficiency between cells and replicates. A novel dual color fluorescent reporter system to assay the promoter activity of a genomic DNA fragment of interest was established by cloning a Discosoma red fluorescent protein gene and a green fluorescent protein gene into a single vector, giving a system in which the ratio between red and green fluorescence is proportional to promoter activity. This system allows real time quantitative monitoring of promoter activity. We validated this approach by assaying the cis-acting regulatory potential of the peroxisome proliferators-activated receptor gamma2 gene.

Inheritance and expression stability of red fluorescent protein gene in transgenic (Tanichthys albonubes)

The stability of the red fluorescent protein(RFP) transgenes is important for application of the transgenic Tanichthys albonubes line.This paper investigated comparatively the inheritance and expression of RFP gene in different transgenic T.albonubes generations.Under the observation of fluorescence microscope,RFP gene was expressed in almost all body tissues,and both F 6 and F 10 generations had a similar expression pattern.Results of self-cross and test-cross breeding experiments of transgenic F 6 and F 10 individuals indicated that segregation of RFP gene still followed Mendelian single-gene inheritance,and their transgenic progenies had a uniform phenotype.The regions including myosin light polypeptide 2 promoter,RFP gene coding sequence and transgene flanking junctions were amplified by PCR from genomic DNA of F 2,F 6 and F 10 generations.Sequence alignment analysis showed that these important regions were well preserved in transgenic fish,and any kind of alteration at nucleotide level was not found.These results suggested that,RFP gene in transgenic T.albonubes maintained stable inheritance and expression through successive generations.

Alternative splicing, expression patterns and promoter characters of vasa-like gene from the silkworm, Bombyx mori

VASA is considered to be one of the most reliable molecular marker of germ cells. In order to study the Bombyx mori vasa-like gene (Bmvlg), the cDNAs of Bmvlg were cloned and sequenced, and the results showed that the Bmvlg gene from the fifth instar larval testes had four alternative splicing isoforms. The open reading frame (ORF) of the longest isoform was composed of 1,806 nucleotides encoding 601 amino acid residues and contained some known conserved domains. The other three isoforms had complete ORF, suggesting that the Bmvlg gene had several alternative splicing forms, completely different from that of Drosophila melanogaster. The results of sequencing demonstrated that the Bmvlg gene promoter had several elements conserved in eukaryotic and gonadal tissue-specific promoters. To detect the specificity of the Bmvlg promoter, a transient expression vector pSK-vlg-DsRed-polyA with a red fluorescent protein gene (DsRed), controlled by the Bmvlg promoter and a vector pIZT/V5-His-vlg-DsRed containing a Bmvlg fused with DsRed driven by the Bmvlg promoter, was constructed, respectively. Red fluorescence could be observed in some transfected BmN cells derived from silkworm ovaries and in the eggs injected with the vector pSK-vlg-DsRed-polyA, but red fluorescence could not be detected in the tissues of silkworm larva, after the transient expression vector was injected into blood, suggesting the Bmvlg promoter had gonadal tissue specificity. The transcription levels of Bmvlg in gonads of the fourth and fifth instar larvae were determined by fluorescent quantitative PCR, and the results revealed that the expression level of the Bmvlg gene in testes was slightly higher than that in ovaries. The expression levels of Bmvlg were lower in the fourth instar larva than that in the fifth instar larvae. Moreover, subcellular localization experiments showed that Bmvlg mainly existed in cytoplasm. These results provided new clues for understanding the function of the Bmvlg gene.

33 IN VITRO IMMUNOGENICITY OF SOMATIC CELL NUCLEAR TRANSFER-DERIVED TRANSGENIC CLONED DOGS

Histocompatible tissue has been generated by somatic cell nuclear transfer (SCNT) and the resultant tissues were not rejected by the immune system of the nucleus donors. In addition, many transgenic animals combined with SCNT have been produced. However, in vitro immunogenicity of transgenic cloned animals originated from the same donor cell with nontransgenic cloned animals has not been assessed until now. The objective of this study was to evaluate the in vitro immunogenicity of cloned dogs with each other, between cloned dogs and transgenic cloned dogs and between transgenic cloned dogs with each other by mixed lymphocyte reaction. In this study, we used cloned beagles (BG1, 2) derived from SCNT using fetal fibroblasts (BF3). Serially, 4 transgenic cloned beagles (Ruppy 1–3, 5) were also genetically engineered from the same donor cell, BF3, with red fluorescent protein (RFP) gene inserted into their genome. We used 2 age-matched healthy female beagle dogs as control dogs. They have different 3 DLA types with all cloned dogs. Peripheral blood mononuclear cells (PBMC) of 2 cloned beagles and 4 transgenic cloned beagles were isolated from whole bloods using Ficoll gradient solution. PBMC from each dog were mixed to auto PBMC, other transgenic cloned dogs and non-related control dogs under the experimental designs. All the mixtures were incubated at 37°C for 4 days, adding BrdU labeling reagent and re-incubated for 24 h. Results are expressed in absorbance mean value ± standard deviation of 450-nm wavelength read by microplate reader. Each cell combination was assayed in 8 replicates. In Experiment 1, PBMC of cloned beagles were combined with equal concentrations of another cloned beagle's PBMC. In Experiment 2, PBMC suspension of Ruppy 1–3, 5 were mixed with equal concentrations of another transgenic cloned beagle's PBMC suspension. In Experiment 3, PBMC suspensions of cloned beagles were mixed with PBMC suspensions of transgenic cloned beagles and reverse reaction was performed. Statistical analysis was performed by using Mann-Whitney U test. In Experiment 1, whereas the absorbance value of mixture of cloned dogs and control dogs shows apparent proliferation, auto mixture of each dog and allo-mixture of BG1 and BG2 show no proliferation (Table 1), indicating immunological factors exposed to PBMC in 2 cloned dogs were compatible. In Experiment 2 among transgenic cloned dogs, no evidence of proliferations in mixed allo-PBMC was shown (Table 1), suggesting in vitro immunogenicity between transgenic cloned dogs was also not shown. In Experiment 3 among cloned dogs and transgenic cloned dogs, no significant difference was found (Table 1). In conclusion, cloned dogs derived from SCNT shared immunological phenotype. Next, immunogenicity among transgenic cloned beagle dogs was not shown despite random insertion of a foreign gene. Lastly, cloned beagles and transgenic cloned beagles show lymphocyte antigen compatibility irrespective of having a foreign gene or not. Table 1.The absorbance values of mixed lymphocytes of 4 transgenic cloned dogs and 2 cloned dogs This study was supported by RNL BIO (#0468-20110001), IPET, MKE (#10033839-2011-13) and Natural Balance Korea.

Selective Targeting of Genetically Engineered Mesenchymal Stem Cells to Tumor Stroma Microenvironments Using Tissue-Specific Suicide Gene Expression Suppresses Growth of Hepatocellular Carcinoma

The use of engineered mesenchymal stem cells (MSCs) as therapeutic vehicles for the treatment of experimental pancreatic and breast cancer has been previously demonstrated. The potential application of MSCs for the treatment of hepatocellular carcinoma (HCC) has been controversial. The general approach uses engineered MSCs to target different aspects of tumor biology, including angiogenesis or the fibroblast-like stromal compartment, through the use of tissue-specific expression of therapeutic transgenes. The aim of the present study was (1) to evaluate the effect of exogenously added MSCs on the growth of HCC and (2) the establishment of an MSC-based suicide gene therapy for experimental HCC. Mesenchymal stem cells were isolated from bone marrow of C57/Bl6 p53(-/-) mice. The cells were injected into mice with HCC xenografts and the effect on tumor proliferation and angiogenesis was evaluated. The cells were then stably transfected with red fluorescent protein (RFP) or Herpes simplex virus thymidine kinase (HSV-Tk) gene under control of the Tie2 promoter/enhancer or the CCL5 promoter. Mesenchymal stem cells were injected intravenously into mice with orthotopically growing xenografts of HCC and treated with ganciclovir (GCV). Ex vivo examination of hepatic tumors revealed tumor-specific recruitment, enhanced tumor growth, and increased microvessel density after nontherapeutic MSC injections. After their homing to the hepatic xenografts, engineered MSCs demonstrated activation of the Tie2 or CCL5 promoter as shown by RFP expression. Application of CCL5/HSV-TK transfected MSCs in combination with GCV significantly reduced tumor growth by 56.4% as compared with the control group and by 71.6% as compared with nontherapeutic MSC injections. CCL5/HSV-TK(+) transfected MSCs proved more potent in tumor inhibition as compared with Tie2/HSV-TK(+) MSCs. Exogenously added MSCs are recruited to growing HCC xenografts with concomitant activation of the CCL5 or Tie2 promoters within the MSCs. Stem cell-mediated introduction of suicide genes into the tumor followed by prodrug administration was effective for treatment of experimental HCC and thus may help fill the existing gap in bridging therapies for patients suffering from advanced HCCs.

Time-Lapse Imaging Reveals Symmetric Neurogenic Cell Division of GFAP-Expressing Progenitors for Expansion of Postnatal Dentate Granule Neurons

Granule cells in the hippocampus, a region critical for memory and learning, are generated mainly during the early postnatal period but neurogenesis continues in adulthood. Postnatal neuronal production is carried out by primary progenitors that express glial fibrillary acidic protein (GFAP) and they are assumed to function as stem cells. A central question regarding postnatal dentate neurogenesis is how astrocyte-like progenitors produce neurons. To reveal cell division patterns and the process of neuronal differentiation of astrocyte-like neural progenitors, we performed time-lapse imaging in cultured hippocampal slices from early postnatal transgenic mice with mouse GFAP promoter-controlled enhanced green fluorescent protein (mGFAP-eGFP Tg mice) in combination with a retrovirus carrying a red fluorescent protein gene. Our results showed that the majority of GFAP-eGFP+ progenitor cells that express GFAP, Sox2 and nestin divided symmetrically to produce pairs of GFAP+ cells (45%) or pairs of neuron-committed cells (45%), whereas a minority divided asymmetrically to generate GFAP+ cells and neuron-committed cells (10%). The present results suggest that a substantial number of GFAP-expressing progenitors functions as transient amplifying progenitors, at least in an early postnatal dentate gyrus, although a small population appears to be stem cell-like progenitors. From the present data, we discuss possible cell division patterns of adult GFAP+ progenitors.

Partial transfection of cells using laminar flows in microchannels

This manuscript describes a convenient method for partial transfection using a Y-shaped microchannel polydimethylsiloxane (PDMS)-glass chip and on-chip cationic lipid-mediated transfection. Enhanced green fluorescent protein genes (pEGFP-N2) were introduced into the COS-7 cells cultured in half of the channel, while red fluorescent protein genes (pDsRed-N1) were introduced into the cells cultured in another half of the channel. This on-chip partial transfection technique provides an avenue for the spatial control of transfection. It is possible to use this technique to perform parallel transfection on chips in order to study cell behaviors under two or more gene transfections in the same culture.