EGFP Marker Gene Research Articles

1012. Survivin-Responsive Conditionally Replicating Adenovirus Exhibits Strictly Cancer-Specific and Efficient Viral Replication

Top of pageAbstract Background, aims and methods: Although conditionally replicating adenovirus (CRA), which shows cancer-selective viral replication and death, is potentially an innovative anti-cancer agent, a current limitation results from the incompleteness of cancer-specific and efficient viral replication. Most importantly, previously reported tissue-specific promoters have the disadvantage of targeting only limited types of cancer, as well as showing less cancer specificity and weak activity even in cancer cells. Here we constructed survivin-responsive CRAs (Surv.CRAs), in which the expression of wild or mutant adenoviral early region 1A gene is regulated by the promoter of survivin, a new member of the inhibitor of apoptosis gene family (Surv.CRAwt and Surv.CRAmt, respectively). Both types of Surv.CRAs have the deletion of E1B55kD, which are driven by the CMV promoter, and contain EGFP marker genes. We carefully explored the cancer-specificity and the effectiveness of viral replication of Surv.CRAs and their therapeutic potential against cancer. Results: Survivin mRNA was expressed in all of the 11 human cancer cells that were derived from various tissue origins, whereas survivin mRNA was very low in normal cells. The survivin promoter was revealed to be strongly active in all these cancer cells studied at levels similar to or even higher than those of representative strong promoters (the RSV and the CMV promoters); in contrast, no detectable activity was observed in normal cells. In careful in vitro experiments (e.g., the speed of the viral replication assessed by the spread of EGFP-positive cells on flowcytometric analysis and by the spread of the cells showing the features of cytopathic effects), both types of Surv.CRAs (i.e., Surv.CRAwt and Surv.CRAmt) efficiently replicated and potently induced death in most types of cancer, in contrast to minimal viral replication resulting in no detectable cytotoxicity in normal cells. A single injection of either of Surv.CRAs into a pre-established tumor expressing survivin, even at a relatively low level, significantly induced tumor death and inhibited tumor growth. There are no different phenotypes in any cells between Sruv.CRAwt and Surv.CRAmt. Furthermore, Surv.CRAs were superior to telomerase (TERT)-dependent CRAs (Tert.CRAs), currently among the best CRAs, both in terms of cancer-specificity and efficiency. Namely, the activity of the survivin promoter was higher than that of the TERT promoter in cancer cells, however, the former is lower than the latter in normal cells. Moreover, Surv.CRAwt is more efficient in replicating in cancer cells than Tert.CRAwt, although the former is more quiescent in normal cells than the latter. Conclusion: Surv.CRAs may be an attractive anti-cancer agent that effectively and specifically treats a variety of cancers.

3. Effects of the cHS4 Insulator Elements on Transgene Expression and Biological Titer of Lentiviral Vector

Modification of retroviral vector to minimize the chance of insertional mutagenesis is the urgent require for stem cell gene therapy. A possible approach is the use of an insulator element identified in the 1.2 Kb fragment from chicken |[beta]|-globin hypersensitivity site 4 (cHS4). When the gene is flanked by the element, both enhancer blocking effect and stable gene expression independent of chromosomal positional effect are observed. One of the problems of the insulator element in the case of HIV-1 vector is the decrease in biological titer, which is thought to be attributed to the increase in the vector RNA size (Blood 101: 4717|[ndash]|4724, 2003). The other possible reason is the characteristic of HIV-1 reverse transcriptase (RT). To form DNA flap during reverse transcription, HIV-1 RT aborts at central termination sequence when it is already double stranded. Retroviral RT have to go through already double stranded LTR for successful reverse transcription, therefore HIV-1 LTR can be a weak point when it is modified. To test the hypothesis, we constructed a series of HIV-1 based vectors. The parent vector carries the EGFP marker gene under the control of U3 from MSCV oncoretroviral vector. The 1.2 Kb cHS4 element or the 0.5 Kb core insulator element was inserted into the parent vector at various positions with either orientation. The relative orientation of cHS4 element against |[beta]|-globin genes and INS1R vector genome is identical. The values shown are the relative biological titer measured on HeLa cells (TU/ml) and the relative biological titer normalized using p24 level measured by ELISA (TU/ng) against those of parental vector, and these are average of four independent experiments (Exp.1) or three independent experiments (Exp.2). The actual titers of parental vector are 2.7|[times]|107 TU/ml and 7.6|[times]|104 TU/ng in Exp.1, and 1.9|[times]|107 TU/ml and 4.0|[times]|104 TU/ng in Exp.2.

Mirk/dyrk1B Decreases the Nuclear Accumulation of Class II Histone Deacetylases during Skeletal Muscle Differentiation

Mirk/dyrk1B is a member of the dyrk/minibrain family of serine/threonine kinases that mediate the transition from growth to differentiation in lower eukaryotes and mammals. Depletion of endogenous Mirk from C2C12 myoblasts by RNA interference blocks skeletal muscle differentiation (Deng, X., Ewton, D., Pawlikowski, B., Maimone, M., and Friedman, E. (2003) J. Biol. Chem. 278, 41347-41354). We now demonstrate that knockdown of Mirk blocks transcription of the muscle regulatory factor myogenin. Co-expression of Mirk with MEF2C, but not MyoD or Myf5, enhanced activation of the myogenin promoter in a Mirk kinase-dependent manner. Mirk activated MEF2 not through direct phosphorylation of MEF2 but by phosphorylation of its inhibitors, the class II histone deacetylases (HDACs). MEF2 is sequestered by class II HDACs such as HDAC5 and MEF2-interacting transcriptional repressor (MITR). Mirk antagonized the inhibition of MEF2C by MITR, whereas kinase-inactive Mirk was ineffective. Mirk phosphorylates class II HDACs at a conserved site within the nuclear localization region, reducing their nuclear accumulation in a dose-dependent and kinase-dependent manner. Moreover, less mutant MITR phosphomimetic at the Mirk phosphorylation site localized in the nucleus than wild-type MITR. Regulation of class II HDACs occurs by multiple mechanisms. Others have shown that calcium signaling leads to phosphorylation of HDACs at 14-3-3-binding sites, blocking their association with MEF2 within the nucleus. Mirk provides another level of regulation. Mirk is induced within the initial 24 h of myogenic differentiation and enables MEF2 to transcribe the myogenin gene by decreasing the nuclear accumulation of class II HDACs.

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15-30% of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.

583. Efficient Non-Viral Gene Delivery to Primary, Leukemia B Cells by mRNA Transfection

Top of pageAbstract There is considerable interest in the use of genetically modified activated or malignant B cells as antigen presenting cells for immunotherapy applications. However, these cells are difficult targets for gene transfer by both viral and non-viral methodologies. Under optimal conditions, we previously demonstrated approximately 50% transfection efficiency by electroporating chronic lymphocytic leukemia (CLL) patients' malignant B cells with DNA plasmid encoding the eGFP marker gene. We next investigated whether transgene expression could be further increased by transfecting mRNA reasoning that in vitro transcribed RNA can be translated simultaneously when it is delivered to the cytosol. Here, we report that transfecting CLL-B cells with mRNA resulted in superior transgene expression. We first electroporated CLL -B cells with 5′-end capped mRNA encoding for the marker gene, eGFP, which was obtained by in vitro transcription of the full length cDNA (on pCI backbone) with a commercially available T7 polymerase kit (Ambion). The transfected cells were analyzed by FACS for transfection efficiency measuring eGFP expression and for cell viability by PI exclusion. Data showed that both cell viability and transfection efficiency were 90% at 3 hr post transfection. CLL patients' malignant B are ineffective antigen-presenting cells, due to the decreased cell surface expression of the co-stimulatory molecules needed to activate T cells after the engagement of T-cell receptor. Forced expression of hCD40L has been shown to up-regulate the expression of co-stimulatory and adhesion molecules and render them to be more efficient antigen-presenting cells, however, its expression has been difficult to achieve in human CLL-B cells. Full length hCD40L mRNA was obtained by the same procedure as described for eGFP and electroporated into CLL-B cells. The transfected cells were immunostained using a FITC-conjugated monoclonal antibody to hCD40L (BD Pharmingen) and analyzed by FACS. The cell surface expression of hCD40L was achieved in greater than 50% of the CLL-B cells as early as 2 hrs post transfection and persisted for at least 48 hrs. Cell viability, when normalized against the control cells was 90%. FACS analysis of the co-stimulatory molecules revealed that hCD40L expression correlated with an up-regulation of both CD80/CD86. Significant up-regulation of CD86 was detected as early as 2 – 4 hr post transfection. The mean fluorescence intensity of CD86 expression was increased approximately 10 fold vs. control cells. mRNA transfection eliminates the concern of genomic modification, gives rise to superior gene expression, safety and cell viability, and offers significant advantages over other gene transfer methodology. Transient gene expression by mRNA transfection should be sufficient and possibly preferable for both in vivo and ex vivo (CTL generation, adoptive) immunotherapy. A scalable, electroporation based system has been developed which will be a useful tool for clinical applications that require large volume mRNA transfection.

Progress towards the development of a transgenic strain of the Australian sheep blowfly ( Lucilia cuprina) suitable for a male-only sterile release program

The Australian sheep blowfly Lucilia cuprina is the most important pest species involved in cutaneous myiasis (flystrike) of sheep in Australia and New Zealand. In New Zealand L. cuprina is primarily controlled through the application of insecticides. However, there is an increased interest in biological methods of control of this species. We have proposed to develop a genetically modified strain of L. cuprina that would be ideal for a male-only sterile release program. To that end we have developed a method for making transgenic L. cuprina using a piggyBac vector and an EGFP marker gene. We have also developed in Drosophila melanogaster a 2-component genetic system for controlling female viability. Females carrying both components of the system die unless fed a diet that contains tetracycline. We anticipate that the female-killing system will need to be optimised for L. cuprina in order to make a strain with the properties required for a male-only release program.

Human foamy virus integrase fails to catalyse the integration of a circular DNA molecule containing an LTR junction sequence.

The presence of closed circular forms of the linear DNA genome of human foamy virus (HFV) has not been established. The ability of the HFV integrase (IN) to catalyse the integration of these circular forms (termed 2 long terminal repeat (LTR) circles) was investigated, with a view to producing a novel hybrid vector. To this end, a construct was made containing, in addition to the enhanced green fluorescent protein (eGFP) marker gene, the last 27 bp of the 3' U5 LTR region of HFV fused to the first 28 bp of the 5' U3 LTR, the latter representing a 2LTR circle. Marker gene expression following transfection of both 293 and 293T cells indicated that the level of integration was not significantly increased by the HFV IN. Moreover, correctly integrated provirus-like forms of the input plasmid could not be detected by PCR. Taken together, these results show that the HFV IN is not able to integrate a circular molecule containing an LTR junction and, hence, the technique is not exploitable as a tool to produce hybrid vectors for gene therapy.

Germ‐line transformation of the Australian sheep blowfly Lucilia cuprina

The Australian sheep blowfly, Lucilia cuprina, is the most important economic insect pest for the sheep industries in Australia and New Zealand. piggyBac-mediated germ-line transformation of L. cuprina was achieved with a helper plasmid that had the Drosophila melanogaster hsp70 promoter controlling expression of the transposase and a piggyBac vector with an EGFP marker gene. Two transformant lines were obtained, at a frequency of approximately 1-2% per fertile G0. One of these lines has a single copy of the transgene, the other most likely has four copies. This is the first report of germ-line transformation of L. cuprina and is an important step towards the generation of engineered strains that would be suitable for male-only release eradication/suppression programmes.

Simple and efficient vectors for retrofitting BACs and PACs with mammalian neoR and EGFP marker genes

Bacterial artificial chromosomes (BACs) and P1 artificial chromosomes (PACs) are widely used to investigate the functions of genes and genomes in mammalian cells invitro and invivo. We have developed a series of vectors which can simply and efficiently be retrofitted onto BACs or PACs. These vectors carry a neoR gene for selection in cells in tissue culture, including ES cells, and also an EGFP gene driven by the strong CAG promoter for quick detection of the DNA in cells. All the plasmids are retrofitted using the loxP site and Cre recombinase and some carry the gamma origin of plasmid R6K which does not function in commonly used bacteria such as DH10B. Retrofitting of PACs and BACs carrying alphoid DNA was very efficient with almost no rearrangement of the highly repetitive alphoid DNA. Following transfer into HT1080 cells and mouse oocytes in tissue culture the DNA could easily be monitored by the EGFP fluorescence.