SV40 Promoter Research Articles

Abstract A65: Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform

Abstract Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also known as 611CTF) is a hyperactive truncated ERBB2 isoform capable of increasing cell migration and invasion in multiple cell types in vitro, and induction of human breast epithelial cell (HMLE) xenograft formation in vivo (Ward et al., Oncogene 2013;32(19):2463-74). p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, though the mechanism to account for such cap-independent p110 t-ERBB2 translation is not well studied. mRNA structural elements termed internal ribosome entry sites (IRESs) can initiate translation in a cap-independent manner when canonical cap-dependent translation is severely compromised. It has been previously demonstrated that the human EGFR 5’ untranslated region (UTR) sequence can initiate the expression of a downstream open reading frame via an IRES (Webb TE, et al., Oncogenesis 2015;4:e134). Therefore, we sought to identify presence of a putative IRES within the 5′ UTR that might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis. Methods: The ERBB2 mRNA 5 ′ UTR from 2 published human ERBB2 transcripts, and several overlapping sequences from full-length ERBB2 start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and promoter-less vector pRFΔP (SV40 promoter removed by restriction digestion), then the resultant constructs were transiently transfected into different cell lines (BT474, SK-BR3, MCF-7, HeLa, CHO). Three control constructs pRF (empty vector control), pRF-Tub (negative control, containing β-tubulin 5 ′ UTR, which lacks IRES activity), and pRF-myc (positive control, containing the well-characterized c-myc IRES) were parallel-transfected. Luciferase expression was then quantified using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer’s instructions. Parallel Western blot analysis and qRT-PCR were also conducted. Results: In this report, we demonstrate that in BT474 and SK-BR3 cells, no IRES activity was detected within human ERBB2 5 ′ UTR sequences under nonstressed conditions, or under serum-starvation, hypoxic conditions, or thapsgargin-induced endoplasmic reticulum stress—conditions when global translation was compromised. The construct pRF-+265/+1561 (within ERBB2 mRNA coding sequence, 5 ′ to the p110 t-ERBB2 start codon) displayed a 10-21 fold increase in firefly/Renilla activity when compared with the empty control pRF and negative control pRF-Tub, consistent with the possibility that the region between +265/+1561 may contain a cryptic promoter. Conclusions: These data are inconsistent with the hypothesis that a 5 ′ UTR IRES-mediated mechanism is involved in the translation of p110 t-ERBB2 isoform, and that other mechanisms are operative in alternative translational regulation/biosynthesis of p110 t-ERBB2 isoform. Citation Format: Yu Zong, Yulin Li, Xiaofei Liu, Mark Daniel Pegram. Dicistronic reporter screen for Internal Ribosome Entry Site (IRES)-mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A65.

A Low-Frequency Amerindian Specific Variant in PALD1, a Negative Regulator of Insulin Signaling, Associates with Type 2 Diabetes

We recently completed a genome wide association study for type 2 diabetes (T2D) in 7659 American Indians who are predominantly of Pima Indian heritage using a custom designed Axiom array. A novel intronic variant (hg19 chr10:72276664 G>C) in PALD1 was identified that associated with T2D (risk allele frequency (RAF)=0.01, OR=2.19[1.49-3.22] per risk allele [C], P=6.6×10-5 adjusted for age, sex, birthyear, PCs 1-5 and accounting for genetic relatedness). Further analysis in a dataset of Urban American Indians (N=3029) representing several tribal groups living in the Phoenix area, identified a directionally consistent association (OR=2.32, P=0.12), but this variant was rare among the entire Urban American Indian sample (RAF=0.003). A meta-analysis of both datasets resulted in a summary OR of 2.15[1.49-3.13], P=4.9×10-5. In Pima Indians, this variant is highly concordant (r2=0.92) with a novel missense variant (hg19 chr10:72298980 C>T, p.[P568S]) in PALD1. The missense variant also had a significant association with T2D in Pima Indians (RAF=0.02, OR=1.80[1.25-2.60], P=0.002) and in Urban American Indians (RAF=0.008, OR=2.29[1.03-5.12], P=0.04). However, after conditional analysis, only the intronic variant remains significant in the Pima Indian dataset. PALD1 is a predicted phosphatase and acts as a negative regulator of insulin signaling. Overexpression of PALD1 leads to lower insulin stimulated AKT phosphorylation and insulin receptor abundance. For preliminary functional studies, a short fragment (100bp) containing each allele of the PALD1 intronic variant (C or G) was cloned upstream of a SV40 promoter luciferase vector and transfected into C2C12 myoblasts. The T2D risk allele (C) fragment had significantly (P=0.004) higher SV40 promoter mediated luciferase activity as compared to the non-risk (G) allele. This result is consistent with the possible role of PALD1 in T2D pathogenesis. Further mechanistic studies are currently ongoing. Disclosure A.K. Nair: None. J.R. Sutherland: None. G.R. Elson: None. P. Piaggi: None. S. Kobes: None. R.L. Hanson: None. C. Bogardus: None. L. Baier: None.

Evaluation of Teneligliptin Effects on Transcriptional Activity of PPARγ in Cell-Based Assays.

The antidiabetic drug teneligliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor with a thiazolidine-specific structure. This study aimed to investigate whether teneligliptin can activate PPARγ directly and/or indirectly in cell-based assays. Promoter assays using the reporter construct driven under the control of the SV40 promoter and the PPAR response element (PPRE) were performed. Luciferase activity was measured after a 3-day incubation of vector-transduced cells with various concentrations of teneligliptin. Treatment of the cells with 50 μM teneligliptin significantly transactivated a reporter gene. The presence of the PPARγ antagonist, GW9662, did not affect the activation of PPRE-reporter expression by teneligliptin. We found that teneligliptin could increase PPARγ activity in cell-based assays irrespective of the PPARγ ligand-binding domain.

Abstract P6-05-05: Discistronic reporter screen for internal ribosome entry site (IRES) - mediated translational regulation of truncated p110 ERBB2 isoform

Abstract Background: We and others demonstrated that truncated p110 ERBB2 (p110 t-ERBB2, also as 611CTF) is a hyperactive truncated ERBB2 isoform capable of increasing cell migration and invasion in multiple cell types in vitro, and induction of human breast epithelial cell (HMLE) xenograft formation in vivo [Ward, et al., Oncogene (2013) 32, 2463–2474]. p110 t-ERBB2 arises through alternative initiation of translation from methionine 611, however, it is unclear how regulation of its expression may be achieved. mRNA structural elements termed internal ribosome entry sites (IRESs) can initiate translation in a cap-independent manner when canonical cap-dependent translation is severely compromised. By cloning the EGFR 5' untranslated region (UTR) between the Renilla and firefly luciferase open reading frames of pRF, Webb, et al. have reported human EGFR 5' UTR sequence can initiate expression of a downstream open reading frame via an IRES [Oncogenesis (2014) 3, e134]. Therefore, we sought to identify presence of a putative IRES within the 5'UTR ERBB2 mRNA which might mediate alternative ERBB2 protein translation initiation under stress conditions and promote p110 t-ERBB2 biosynthesis. Methods: Discistronic reporter pRF was used as the backbone vector to detect IRES activity. Promoter-less vector pRFΔP were constructed by removing SV40 promoter via restriction digestion. The HER2 mRNA 5'UTR (from both variant 1 and variant 3) and several overlapping sequences from full length ERBB2 (p185 ERBB2) start codon to p110 t-ERBB2 start codon were cloned between the Renilla and firefly luciferase open reading frames of pRF and pRFΔP, then the resultant constructswere transiently transfected into different cell lines(BT474, SK-BR3, MCF-7, HeLa, CHO). Three control constructs pRF (empty vector control), pRF-Tub (negative control, containing βtubulin 5'UTR, which lacks IRES activity) and pRF-myc (positive control, containing the well-characterized c-myc IRES) were parallel-transfected. Luciferase expression was then quantified using a Dual Luciferase Assay Kit (Promega, Madison, WI, USA) following manufacturer's instructions. Parallel western blot analysis and qRT-PCR were also conducted. Results: In this report, we demonstrate that in BT474 and SK-BR3 cells, no IRES activity was detected within human ERBB2 5'UTR sequences under non-stressed conditions, or under serum-starvation, hypoxic conditions or thapsgargin-induced endoplasmic reticulum stress -- conditions when global translation was compromised.The construct pRF-+265/+1561 (within ERBB2 mRNA coding sequence, 5' to the p110 t-ERBB2 start codon) displayed a 10-21 fold increase in firefly/Renilla activity when compared with the empty control pRF and negative control pRF-Tub,consistent with the possibility that the region between +265/+1561 may contain a cryptic promoter. Conclusions: These data are inconsistent with the hypothesis that a 5'UTR IRES-mediated mechanism is involved in the translation of p110 t-ERBB2 isoform, and that other mechanisms are operative in alternative translational regulation/biosynthesis of p110 ERBB2 isoform. Citation Format: Zong Y, Li Y, Liu X, Pegram MD. Discistronic reporter screen for internal ribosome entry site (IRES) - mediated translational regulation of truncated p110 ERBB2 isoform [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-05-05.

Matrix attachment region combinations increase transgene expression in transfected Chinese hamster ovary cells

Matrix attachment regions (MARs) are cis-acting DNA elements that can increase transgene expression levels in a CHO cell expression system. To investigate the effects of MAR combinations on transgene expression and the underlying regulatory mechanisms, we generated constructs in which the enhanced green fluorescent protein (eGFP) gene flanked by different combinations of human β-interferon and β-globin MAR (iMAR and gMAR, respectively), which was driven by the cytomegalovirus (CMV) or simian virus (SV) 40 promoter. These were transfected into CHO-K1 cells, which were screened with geneticin; eGFP expression was detected by flow cytometry. The presence of MAR elements increased transfection efficiency and transient and stably expression of eGFP expression under both promoters; the level was higher when the two MARs differed (i.e., iMAR and gMAR) under the CMV but not the SV40 promoter. For the latter, two gMARs showed the highest activity. We also found that MARs increased the ratio of stably transfected positive colonies. These results indicate that combining the CMV promoter with two different MAR elements or the SV40 promoter with two gMARs is effective for inducing high expression level and stability of transgenes.

Transient expression of the enhanced green fluorescence protein (egfp) gene in Tetraselmis subcordiformis (Chlorodendrales, Chlorophyta) with three exogenous promoters

:A stable transformation system for Tetraselmis subcordiformis had been established, but a low transformation rate limits its application to this alga. As a promoter is a key factor for transgenic research, a suitable promoter could increase the transformation rate. To improve a transformation system for T. subcordiformis, three commonly used promoters for algal transformation were selected. The CMV, CaMV35S and SV40 promoters were transferred to T. subcordiformis by glass-bead agitation and particle bombardment, with the enhanced green fluorescence protein (egfp) gene as the reporter gene. The transformation rate was calculated according to the GFP fluorescence in transformed algae. Results showed that the CaMV 35S promoter transformation efficiency was higher than that of CMV and SV40 promoters. This report will contribute to efforts to establish a high-efficiency transformation system and expands the fundamental research and applications of this alga.

Establishment of a HEK293T cell line able to site-specifically integrate and stably express GDNF by rAAV-2 vector

Background: Using recombinant adeno-associated virus 2 (rAAV-2), we attempted to establish a HEK293T cell line that is able to site-specifically integrate and stably express glial cell line-derived neurotrophic factor (GDNF). Results: Recombinant vector with enhanced green fluorescent protein (EGFP) and GDNF (pTR-P5-EGFP-IRES-GDNF), as well as that carrying Rep genes and SV40 promoters (pSVAV2) were constructed and packed. HEK293T cells were co-infected with rAAV-2/EGFP-GDNF and rAAV-2/SVAV2 virus separately at 1 × 10 4 , 1 × 10 5 , and 1 × 10 6 of multiplicity of infection (MOI). The efficiency of transduction was detected using flow cytometry. Additionally, the infected HEK293T cells were separately validated by touchdown polymerase chain reaction (PCR) and Western-blot. After 72 h of transduction, the rate of EGFP positive cell was 22%, 45% and 49% at the MOIs of 1 × 10 4 , 1 × 10 5 and 1 × 10 6 , respectively. On the 3rd, 6th and 9th day of cell passage, there was no significant difference in the cell viability and proliferation rate between transduction and control groups. Importantly, touchdown PCR showed that there was a specific PCR amplified product band in the lane of infected cells. Furthermore, GDNF expression was detected in the infected cells after 15 and 180 d of cultivation. Conclusions: A HEK293T cell line able to site-specifically integrate and stably express GDNF was established.

Impact of Different Promoters on Episomal Vectors Harbouring Characteristic Motifs of Matrix Attachment Regions.

We previously demonstrated that the characteristic sequence of matrix attachment regions (MARs) allows transgenes to be maintained episomally in CHO cells. In the present study, six commonly used promoters from human cytomegalovirus major immediate-early (CMV), simian vacuolating virus 40 (SV40), Rous sarcoma virus, Homo sapiens ubiquitin C, phosphoglycerate kinase, and β-globin, respectively, were evaluated to determine their effects on transgene expression and stability in CHO cells stably transfected via the episomal vector harbouring characteristic MAR motifs. The CHO cells were transfected with vectors and then screened using G418, after which the stably transfected cells were split into two and further cultured either in the presence or absence of G418. Of the six promoters, the CMV promoter yielded the highest transgene expression levels and the highest transfection efficiency, whereas the SV40 promoter maintained transgene expression more stably during long-term culture than the other promoters did. The CMV and SV40 promoter-containing vectors were furthermore episomally maintained and conferred sustained eGFP expression in the cells even under nonselective conditions. On the basis of these findings, we conclude that the CMV promoter performs best in terms of yielding both high expression levels and high levels of stability using this episomal vector system.

AAV-mediated transduction and targeting of retinal bipolar cells with improved mGluR6 promoters in rodents and primates.

Adeno-associated virus (AAV) vectors have been a powerful gene delivery vehicle to the retina for basic research and gene therapy. For many of these applications, achieving cell-type specific targeting and high transduction efficiency is desired. Recently, there has been increasing interest in AAV-mediated gene targeting to specific retinal bipolar cell types. A 200-bp enhancer in combination with a basal SV40 promoter has been commonly used to target transgenes into ON-type bipolar cells. In the current study, we searched for additional cis-regulatory elements in the mGluR6 gene for improving AAV-mediated transduction efficiency into retinal bipolar cells. Our results showed that the combination of the endogenous mGluR6 promoter with additional enhancers in the introns of the mGluR6 gene markedly enhanced AAV transduction efficiency as well as made the targeting more selective for rod bipolar cells in mice. Furthermore, the AAV vectors with the improved promoter could target to ON bipolar cells with robust transduction efficiency in the para-fovea and the far peripheral retina of marmoset monkeys. The improved mGluR6 promoter constructs could provide a valuable tool for genetic manipulation in rod bipolar cells in mice and facilitate clinical applications for ON bipolar cell-based gene therapies.

Sleeping Beauty transposon-based system for rapid generation of HBV-replicating stable cell lines

The stable HBV-replicating cell lines, which carry replication-competent HBV genome stably integrated into the genome of host cell, are widely used to evaluate the effects of antiviral agents. However, current methods to generate HBV-replicating cell lines, which are mostly dependent on random integration of foreign DNA via plasmid transfection, are less-efficient and time-consuming. To address this issue, we constructed an all-in-one Sleeping Beauty transposon system (denoted pTSMP-HBV vector) for robust generation of stable cell lines carrying replication-competent HBV genome of different genotype. This vector contains a Sleeping Beauty transposon containing HBV 1.3-copy genome with an expression cassette of the SV40 promoter driving red fluorescent protein (mCherry) and self-cleaving P2A peptide linked puromycin resistance gene (PuroR). In addition, a PGK promoter-driven SB100X hyperactive transposase cassette is placed in the outside of the transposon in the same plasmid.The HBV-replicating stable cells could be obtained from pTSMP-HBV transfected HepG2 cells by red fluorescence-activated cell sorting and puromycin resistant cell selection within 4-week. Using this system, we successfully constructed four cell lines carrying replication-competent HBV genome of genotypes A–D. The replication and viral protein expression profiles of these cells were systematically characterized. In conclusion, our study provides a high-efficiency strategy to generate HBV-replicating stable cell lines, which may facilitate HBV-related virological study.

Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells.

Inducible transcription systems play a crucial role in a wide array of synthetic biology circuits. However, the majority of inducible promoters are constructed from a limited set of tried-and-true promoter parts, which are susceptible to common shortcomings such as high basal expression levels (i.e., leakiness). To expand the toolbox for regulated mammalian gene expression and facilitate the construction of mammalian genetic circuits with precise functionality, we quantitatively characterized a panel of eight core promoters, including sequences with mammalian, viral, and synthetic origins. We demonstrate that this selection of core promoters can provide a wide range of basal gene expression levels and achieve a gradient of fold-inductions spanning 2 orders of magnitude. Furthermore, commonly used parts such as minimal CMV and minimal SV40 promoters were shown to achieve robust gene expression upon induction, but also suffer from high levels of leakiness. In contrast, a synthetic promoter, YB_TATA, was shown to combine low basal expression with high transcription rate in the induced state to achieve significantly higher fold-induction ratios compared to all other promoters tested. These behaviors remain consistent when the promoters are coupled to different genetic outputs and different response elements, as well as across different host-cell types and DNA copy numbers. We apply this quantitative understanding of core promoter properties to the successful engineering of human T cells that respond to antigen stimulation via chimeric antigen receptor signaling specifically under hypoxic environments. Results presented in this study can facilitate the design and calibration of future mammalian synthetic biology systems capable of precisely programmed functionality.

A/T gap tolerance in the core sequence and flanking sequence requirements of non-canonical p53 response elements.

The canonical core sequence of the p53 response element, CATG, has a two-base A/T gap. Previously, we found that p53 can also activate a non-canonical four-base A/T gap CATATG core sequence. In this study, we investigated the possible number of A/T bases used by p53 and showed that a six-base A/T gap CATATATG core sequence was the maximum A/T gap in the p53 response element that could be upregulated by p53 and p63. Canonical and non-canonical p53 response elements also have three-base flanking sequences. A/T bases could be substituted by G/C bases, including CACACG and CGTGTG, but not CGCGCG. We found that the SV40 promoter with functional six- and two-base A/T gap core sequences could be activated by TAp63γ and that TAp63γ could upregulate SV40 small and large T antigens expression in COS7 cells. We also found that the distal region of PUMA promoter with functional two six-base A/T gap core sequences could be activated by TAp63γ in 293T cells. These new findings could provide novel rules for the non-canonical p53 family response element and could extend the entire p53 family regulation network.

Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein.

Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity.

Abstract 15800: Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

Microsomal triglyceride transfer protein (MTP) is heterodimeric protein complex consisting of a unique 97 kDa protein with lipid transfer activity and the ubiquitous 58 kDa protein disulfide isomerase. The complex is essential for assembly of triglyceride-rich apolipoprotein (apo) B-containing lipoproteins. In tissues that do not synthesize apoB nor assemble lipoproteins, it is presumed to serve specialized needs in lipid trafficking, such as in the lipidation of CD1d in antigen presenting cells, including adipocytes. Previous studies in our laboratory identified a novel isoform of MTP in mice that we named MTP-B. MTP-B has an alternative first exon (Ex1B) located 2.7 kbp upstream of the first exon (Ex1A) of MTP-A, the canonical isoform of MTP. The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study, we have identified another splice variant, MTP-C, that contains both exons Ex1B and Ex1A. PCR analysis revealed that MTP-C mRNA is expressed in a number of tissues including liver, ovary, heart, kidney, brain and testis. In CHO and HEK 293 cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’ untranslated region (UTR) of MTP-C revealed six ATGs (uATGs) upstream of the translation initiation site for MTP-A, which is the only viable start site in frame with the main open reading frame (ORF), and three potential upstream ORFs (uORFs). Since uATGs and uORFs are generally associated with repressed translational efficiency, we inserted the 5’ UTR of MTP-C into a pGL3 vector between the SV40 promoter and luciferase reporter gene and transfected it into CHO cells. Luciferase activity was significantly reduced (45.5 ± 6.4%) compared to pGL3-Control vector transfected cells. This suggests the reduced protein expression observed in MTP-C transfected cells resulted in part from decreased translation efficiency. We conclude that uORFs in the 5’ UTR of MTP-C repress translation, providing a mechanism to fine tune MTP levels to meet the needs of the cell. uORFs in human MTP-C could represent novel therapeutic targets for regulation of MTP expression and plasma lipid levels.

Human Obesity Associated with an Intronic SNP in the Brain-Derived Neurotrophic Factor Locus.

Brain-derived neurotrophic factor (BDNF) plays a key role in energy balance. In population studies, SNPs of the BDNF locus have been linked to obesity, but the mechanism by which these variants cause weight gain is unknown. Here, we examined human hypothalamic BDNF expression in association with 44 BDNF SNPs. We observed that the minor C allele of rs12291063 is associated with lower human ventromedial hypothalamic BDNF expression (p < 0.001) and greater adiposity in both adult and pediatric cohorts (p values < 0.05). We further demonstrated that the major T allele for rs12291063 possesses a binding capacity for the transcriptional regulator, heterogeneous nuclear ribonucleoprotein D0B, knockdown of which disrupts transactivation by the T allele. Binding and transactivation functions are both disrupted by substituting C for T. These findings provide a rationale for BDNF augmentation as a targeted treatment for obesity in individuals who have the rs12291063 CC genotype.

Intradermal delivery of DNA encoding HCV NS3 and perforin elicits robust cell-mediated immunity in mice and pigs.

Currently, no vaccine is available against hepatitis C virus (HCV), and although DNA vaccines have considerable potential, this has not been realised. Previously, the efficacy of DNA vaccines for human immunodeficiency virus (HIV) and HCV was shown to be enhanced by including the gene for a cytolytic protein, viz. perforin. In this study, we examined the mechanism of cell death by this bicistronic DNA vaccine, which encoded the HCV non-structural protein 3 (NS3) under the control of the CMV promoter and perforin is controlled by the SV40 promoter. Compared with a canonical DNA vaccine and a bicistronic DNA vaccine encoding NS3 and the proapoptotic gene NSP4, the perforin-containing vaccine elicited enhanced cell-mediated immune responses against the NS3 protein in vaccinated mice and pigs, as determined by ELISpot and intracellular cytokine staining, whereas a mouse challenge model suggested that the immunity was CD8(+) T-cell-dependent. The results of the study showed that the inclusion of perforin in the DNA vaccine altered the fate of NS3-positive cells from apoptosis to necrosis, and this resulted in more robust immune responses in mice and pigs, the latter of which represents an accepted large animal model in which to test vaccine efficacy.

Transgene expression in tick cells using Agrobacterium tumefaciens.

Ticks transmit infectious agents to humans and other animals. Genetic manipulation of vectors like ticks could enhance the development of alternative disease control strategies. Transgene expression using the phytopathogen Agrobacterium tumefaciens has been shown to promote the genetic modification of non-plant cells. In the present work we developed T-DNA constructs for A. tumefaciens to mediate transgene expression in HeLa cells as well as Rhipicephalus microplus tick cells. Translational fusions eGfp:eGfp or Salp15:eGfp, including the enhanced-green fluorescent protein and the Ixodes scapularis salivary factor SALP15 genes, were constructed using the CaMV 35S (cauliflower mosaic virus) promoter, "PBm" tick promoter (R. microplus pyrethroid metabolizing esterase gene) or the Simian Virus SV40 promoter. Confocal microscopy, RT-PCR and Western-blot assays demonstrated transgene(s) expression in both cell lines. Transgene expression was also achieved in vivo, in both R. microplus and I. scapularis larvae utilizing a soaking method including the A. tumefaciens donor cells and confirmed by nested-RT-PCR showing eGfp or Salp15 poly-A-mRNA(s). This strategy opens up a new avenue to express exogenous genes in ticks and represents a potential breakthrough for the study of tick-host pathophysiology.

Non-invasive gene targeting to the fetal brain after intravenous administration and transplacental transfer of plasmid DNA using PEGylated immunoliposomes

Research was undertaken to establish transplacental delivery of active genes to fetal brain by a non-viral vector, antibody-specific targeted therapeutic procedure. PEGylated immunoliposomes (PILs) containing firefly luciferase DNA under the influence of the SV40 promoter injected intravenously into near-term pregnant mice produced luminometric evidence of CNS tissue luciferase activity at 48-h post-injection in all newborn pups. In utero delivery of this pGL3 DNA was shown after a single i.v. injection in maternal and neonatal brains, spleen and lesser amounts in lungs, with only negligible background levels in negative controls exposed to unencapsulated pDNA. In addition to studies of normal wild-type mice, we similarly injected pregnant Lafora Knockout (EPM2a null-mutant) and demonstrated luciferase activity days later in the maternal and newborn pup brains of both types. Delivery of PILs containing a second reporter gene (the pSV40 beta-galactosidase transgene) transplacentally by the same procedure was also successful. Histochemical and biochemical demonstration of beta-galactosidase was documented for all mutant and non-mutant neonates. Brain areas of highest Lafora body development (such as the hippocampus and pontine nuclei) showed intraneuronal beta-glucosidase activity. We conclude that receptor-mediated transport of PIL-borne gene therapeutics across both the placental barrier as well as the fetal BBB in utero is feasible.

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1&amp;#945; Promoter Using Gibson Assembly

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.

Generation of plasmid vectors expressing FLAG-tagged proteins under the regulation of human elongation factor-1α promoter using Gibson assembly.

Gibson assembly (GA) cloning offers a rapid, reliable, and flexible alternative to conventional DNA cloning methods. We used GA to create customized plasmids for expression of exogenous genes in mouse embryonic stem cells (mESCs). Expression of exogenous genes under the control of the SV40 or human cytomegalovirus promoters diminishes quickly after transfection into mESCs. A remedy for this diminished expression is to use the human elongation factor-1 alpha (hEF1α) promoter to drive gene expression. Plasmid vectors containing hEF1α are not as widely available as SV40- or CMV-containing plasmids, especially those also containing N-terminal 3xFLAG-tags. The protocol described here is a rapid method to create plasmids expressing FLAG-tagged CstF-64 and CstF-64 mutant under the expressional regulation of the hEF1α promoter. GA uses a blend of DNA exonuclease, DNA polymerase and DNA ligase to make cloning of overlapping ends of DNA fragments possible. Based on the template DNAs we had available, we designed our constructs to be assembled into a single sequence. Our design used four DNA fragments: pcDNA 3.1 vector backbone, hEF1α promoter part 1, hEF1α promoter part 2 (which contained 3xFLAG-tag purchased as a double-stranded synthetic DNA fragment), and either CstF-64 or specific CstF-64 mutant. The sequences of these fragments were uploaded to a primer generation tool to design appropriate PCR primers for generating the DNA fragments. After PCR, DNA fragments were mixed with the vector containing the selective marker and the GA cloning reaction was assembled. Plasmids from individual transformed bacterial colonies were isolated. Initial screen of the plasmids was done by restriction digestion, followed by sequencing. In conclusion, GA allowed us to create customized plasmids for gene expression in 5 days, including construct screens and verification.