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Non-viral transposons have been used successfully for genetic modification of clinically relevant cells including embryonic stem, induced pluripotent stem, hematopoietic stem and primary human T cell types. However, there has been limited evaluation of undesired genomic effects when using transposons for human genome modification. The prevalence of piggyBac(PB)-like terminal repeat (TR) elements in the human genome raises concerns. We evaluated if there were undesired genomic effects of the PB transposon system to modify human cells. Expression of the transposase alone revealed no mobilization of endogenous PB-like sequences in the human genome and no increase in DNA double-strand breaks. The use of PB in a plasmid containing both transposase and transposon greatly increased the probability of transposase integration; however, using transposon and transposase from separate vectors circumvented this. Placing a eGFP transgene within transposon vector backbone allowed isolation of cells free from vector backbone DNA. We confirmed observable directional promoter activity within the 5'TR element of PB but found no significant enhancer effects from the transposon DNA sequence. Long-term culture of primary human cells modified with eGFP-transposons revealed no selective growth advantage of transposon-harboring cells. PB represents a promising vector system for genetic modification of human cells with limited undesired genomic effects.
Published by Oxford University Press on behalf of Nucleic Acids Research 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.
RNAi based strategies to induce gene silencing are commonly employed in numerous model organisms but have not been extensively used in zebrafish. We found that introduction of transgenes containing convergent transcription units in zebrafish embryos induced stable transcriptional gene silencing (TGS) in cis and trans for reporter (mCherry) and endogenous (One-Eyed Pinhead (OEP) and miR-27a/b) genes. Convergent transcription enabled detection of both sense and antisense transcripts and silencing was suppressed upon Dicer knockdown, indicating processing of double stranded RNA. By ChIP analyses, increased silencing was accompanied by enrichment of the heterochromatin mark H3K9me3 in the two convergently arranged promoters and in the intervening reading frame. Our work demonstrates that convergent transcription can induce gene silencing in zebrafish providing another tool to create specific temporal and spatial control of gene expression.
Phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates the function of ion channels and transporters. Here, we demonstrate that PIP2 directly binds the human dopamine (DA) transporter (hDAT), a key regulator of DA homeostasis and a target of the psychostimulant amphetamine (AMPH). This binding occurs through electrostatic interactions with positively charged hDAT N-terminal residues and is shown to facilitate AMPH-induced, DAT-mediated DA efflux and the psychomotor properties of AMPH. Substitution of these residues with uncharged amino acids reduces hDAT-PIP2 interactions and AMPH-induced DA efflux without altering the hDAT physiological function of DA uptake. We evaluated the significance of this interaction in vivo using locomotion as a behavioral assay in Drosophila melanogaster. Expression of mutated hDAT with reduced PIP2 interaction in Drosophila DA neurons impairs AMPH-induced locomotion without altering basal locomotion. We present what is to our knowledge the first demonstration of how PIP2 interactions with a membrane protein can regulate the behaviors of complex organisms.
We previously showed that asunder (asun) is a critical regulator of dynein localization during Drosophila spermatogenesis. Because the expression of asun is much higher in Drosophila ovaries and early embryos than in testes, we herein sought to determine whether ASUN plays roles in oogenesis and/or embryogenesis. We characterized the female germline phenotypes of flies homozygous for a null allele of asun (asun(d93)). We find that asun(d93) females lay very few eggs and contain smaller ovaries with a highly disorganized arrangement of ovarioles in comparison to wild-type females. asun(d93) ovaries also contain a significant number of egg chambers with structural defects. A majority of the eggs laid by asun(d93) females are ventralized to varying degrees, from mild to severe; this ventralization phenotype may be secondary to defective localization of gurken transcripts, a dynein-regulated step, within asun(d93) oocytes. We find that dynein localization is aberrant in asun(d93) oocytes, indicating that ASUN is required for this process in both male and female germ cells. In addition to the loss of gurken mRNA localization, asun(d93) ovaries exhibit defects in other dynein-mediated processes such as migration of nurse cell centrosomes into the oocyte during the early mitotic divisions, maintenance of the oocyte nucleus in the anterior-dorsal region of the oocyte in late-stage egg chambers, and coupling between the oocyte nucleus and centrosomes. Taken together, our data indicate that asun is a critical regulator of dynein localization and dynein-mediated processes during Drosophila oogenesis.
Copyright © 2013 Elsevier Inc. All rights reserved.
β cell failure in type 2 diabetes (T2D) is associated with hyperglycemia, but the mechanisms are not fully understood. Congenital hyperinsulinism caused by glucokinase mutations (GCK-CHI) is associated with β cell replication and apoptosis. Here, we show that genetic activation of β cell glucokinase, initially triggering replication, causes apoptosis associated with DNA double-strand breaks and activation of the tumor suppressor p53. ATP-sensitive potassium channels (KATP channels) and calcineurin mediate this toxic effect. Toxicity of long-term glucokinase overactivity was confirmed by finding late-onset diabetes in older members of a GCK-CHI family. Glucagon-like peptide-1 (GLP-1) mimetic treatment or p53 deletion rescues β cells from glucokinase-induced death, but only GLP-1 analog rescues β cell function. DNA damage and p53 activity in T2D suggest shared mechanisms of β cell failure in hyperglycemia and CHI. Our results reveal membrane depolarization via KATP channels, calcineurin signaling, DNA breaks, and p53 as determinants of β cell glucotoxicity and suggest pharmacological approaches to enhance β cell survival in diabetes.
Copyright © 2014 Elsevier Inc. All rights reserved.
Renal medullary interstitial cells (RMIC) are specialized fibroblast-like cells that exert important functions in maintaining body fluid homeostasis and systemic blood pressure. Here, we generated a RMIC specific tenascin-C promoter driven inducible CreER2 knockin mouse line with an EGFP reporter. Similar as endogenous tenascin-C expression, the reporter EGFP expression in the tenascin-C-CreER2(+/-) mice was observed in the inner medulla of the kidney, and co-localized with COX2 but not with AQP2 or AQP1, suggesting selective expression in RMICs. After recombination (tenascin-C-CreER2(+/-)/ROSA26-lacZ(+/-) mice + tamoxifen), β-gal activity was restricted to the cells in the inner medulla of the kidney, and didn't co-localize with AQP2, consistent with selective Cre recombinase activity in RMICs. Cre activity was not obvious in other major organs or without tamoxifen treatment. This inducible RMIC specific Cre mouse line should therefore provide a novel tool to manipulate genes of interest in RMICs.
Uchl1 encodes the protein gene product 9.5 antigen (PGP9.5) that is a widely used to identify migrating neural progenitors in the PNS, mature neurons of the central and peripheral nervous systems, as well as neuroendocrine cells. To facilitate analysis of developing peripheral neurons, we linked regulatory regions of Uchl1 carried within a 160kb bacterial artificial chromosome (BAC) to the dual fluorescent reporter H2BmCherry:GFP-gpi. The Uchl1-H2BmCherry:GFP-gpi transgene exhibits robust expression and allows clear discrimination of individual cells and cellular processes in cranial ganglia, sympathetic chain, the enteric nervous system (ENS), and autonomic ganglia of the urogenital system. The transgene also labels subsets of cells in endocrine tissues where earlier in situ hybridization (ISH) studies have previously identified expression of this deubiquinating enzyme. The Uchl1-H2BmCherry:GFP-gpi transgene will be a powerful tool for static and live imaging, as well as isolation of viable neural progenitors to investigate processes of autonomic neurogenesis.
Copyright © 2013 Wiley Periodicals, Inc.
Cre/LoxP has broad utility for studying the function, development, and oncogenic transformation of pancreatic cells in mice. Here we provide an overview of the Cre driver lines that are available for such studies. We discuss how variegated expression, transgene silencing, and recombination in undesired cell types have conspired to limit the performance of these lines, sometimes leading to serious experimental concerns. We also discuss preferred strategies for achieving high-fidelity driver lines and remind investigators of the continuing need for caution when interpreting results obtained from any Cre/LoxP-based experiment performed in mice.
Copyright © 2013 Elsevier Inc. All rights reserved.
Transposons permit permanent cellular genome engineering in vivo. However, transgene expression falls rapidly postdelivery due to a variety of mechanisms, including immune responses. We hypothesized that delaying initial transgene expression would improve long-term transgene expression by using an engineered piggyBac transposon system that can regulate expression. We found that a 2-part nonviral Tet-KRAB inducible expression system repressed expression of a luciferase reporter in vitro. However, we also observed nonspecific promoter-independent repression. Thus, to achieve temporary transgene repression after gene delivery in vivo, we utilized a nonintegrating version of the repressor plasmid while the gene of interest was delivered in an integrating piggyBac transposon vector. When we delivered the luciferase transposon and repressor to immunocompetent mice by hydrodynamic injection, initial luciferase expression was repressed by 2 orders of magnitude. When luciferase expression was followed long term in vivo, we found that expression was increased >200-fold compared to mice that received only the luciferase transposon and piggyBac transposase. We found that repression of early transgene expression could prevent the priming of luciferase-specific T cells in vivo. Therefore, transient transgene repression postgene delivery is an effective strategy for inhibiting the antitransgene immune response and improving long-term expression in vivo without using immunosuppression.
The piggyBac transposon system is a promising nonviral method to genetically modify T cells for immunotherapeutic applications. To evaluate the regulation and stability of transgene expression in human T cells modified with piggyBac-transposons, peripheral blood mononuclear cells were nucleofected with transposase and an enhanced green fluorescence protein (eGFP)-expressing transposon. Single-cell clones that were subsequently stimulated and expanded exhibited homogenous eGFP expression for >26 weeks in culture. CD3 stimulation of the T-cell receptor together with CD28-mediated costimulation resulted in an approximate 10-fold transient increase in eGFP expression, but immunomodulatory cytokines, including interferon-γ, interleukin-12, interleukin-4, and transforming growth factor-β, did not alter transgene expression in actively dividing, activated, or resting T cells. Epigenetic modification with 5-azacytidine or trichostatin-A increased transgene expression indicating that piggyBac-mediated transgene expression could be modulated by methylation or histone acetylation, respectively. We performed transposon copy number analysis of populations of stably transfected T cells, comparing transposon plasmids of 5.6 and 3.5 kb. The smaller vector achieved an average of 22 transposon copies per cell, whereas the larger vector achieved 1.6 copies/cell, implying that transposon copy number can be engineered to be low or high depending on the vector used. Our results provide important insight into the ability of piggyBac to achieve stable genetic modification of T cells for immunotherapy applications and how transgene expression might be regulated by TCR activation, cytokines, and epigenetic mechanisms.