Rescue Transgene Research Articles

A DREaMR system to simplify combining mutations with rescue transgenes in Aedes aegypti.

In most experimental animals, it is challenging to combine mutations and rescue transgenes and to use bipartite systems to assess gene expression. To circumvent the difficulties in combining multiple genetic elements, we developed the DREaMR (Drug-on, REporter, Mutant, Rescue) system. Using Drosophila white as the initial model, we demonstrated that introduction of a single insertion by CRISPR/Cas9 created a null mutation, a tagged rescue construct, which could be induced with doxycycline, and which allowed assessment of protein expression. To create a DREaMR in an organism in which combining multiple genetic elements is more problematic than in Drosophila, we tested the mosquito, Aedes aegypti-the insect vector for dengue, yellow fever, Zika, and other viral diseases. We generated a DREaMR allele in the kh gene, which permitted us to induce expression of the rescue construct, and detect expression of Kh. Thus, this system avoids the need to perform genetic crosses to introduce an inducible rescue transgene in a mutant background, or to combine driver and reporter lines to examine expression of the targeted protein. We propose that DREaMR provides a system that can be applied to additional mosquito vectors as well as other organisms in which CRISPR/Cas9 is effective.

Regulation of lipid homeostasis by the TBC protein dTBC1D22 via modulation of the small GTPase Rab40 to facilitate lipophagy.

The regulation of lipid homeostasis is not well understood. Using forward genetic screening, we demonstrate that the loss of dTBC1D22, an essential gene that encodes a Tre2-Bub2-Cdc16 (TBC) domain-containing protein, results in lipid droplet accumulation in multiple tissues. We observe that dTBC1D22 interacts with Rab40 and exhibits GTPase activating protein (GAP) activity. Overexpression of either the GTP- or GDP-binding-mimic form of Rab40 results in lipid droplet accumulation. We observe that Rab40 mutant flies are defective in lipid mobilization. The lipid depletion induced by overexpression of Brummer, a triglyceride lipase, is dependent on Rab40. Rab40 mutant flies exhibit decreased lipophagy and small size of autolysosomal structures, which may be due to the defective Golgi functions. Finally, we demonstrate that Rab40 physically interacts with Lamp1, and Rab40 is required for the distribution of Lamp1 during starvation. We propose that dTBC1D22 functions as a GAP for Rab40 to regulate lipophagy.

Gene drive and resilience through renewal with next generation Cleave and Rescue selfish genetic elements

Gene drive-based strategies for modifying populations face the problem that genes encoding cargo and the drive mechanism are subject to separation, mutational inactivation, and loss of efficacy. Resilience, an ability to respond to these eventualities in ways that restore population modification with functional genes, is needed for long-term success. Here, we show that resilience can be achieved through cycles of population modification with "Cleave and Rescue" (ClvR) selfish genetic elements. ClvR comprises a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene and a recoded version of the essential gene resistant to cleavage. ClvR spreads by creating conditions in which those lacking ClvR die because they lack functional versions of the essential gene. Cycles of modification can, in principle, be carried out if two ClvR elements targeting different essential genes are located at the same genomic position, and one of them, ClvRn+1, carries a Rescue transgene from an earlier element, ClvRnClvRn+1 should spread within a population of ClvRn, while also bringing about a decrease in its frequency. To test this hypothesis, we first show that multiple ClvRs, each targeting a different essential gene, function when located at a common chromosomal position in Drosophila We then show that when several of these also carry the Rescue from a different ClvR, they spread to transgene fixation in populations fixed for the latter and at its expense. Therefore, genetic modifications of populations can be overwritten with new content, providing an ongoing point of control.

Combining CRISPR/Cas9-mediated knockout with genetic complementation for in-depth mechanistic studies in human ES cells.

Gene regulatory networks that control pluripotency of human embryonic stem cells (hESCs) are of considerable interest for regenerative medicine. RNAi and CRISPR/Cas9 technologies have allowed the identification of hESC regulators on a genome-wide scale. However, these technologies are ill-suited for mechanistic studies because knockdown/knockout clones of essential genes do not grow in culture. We have developed a genetic rescue strategy that combines CRISPR/Cas9-mediated knockout with TALEN-mediated integration of a doxycycline-inducible rescue transgene into a constitutive AASV1 locus. The resulting rescue clones are stable in culture, allow modulation of the rescue transgene dosage by titration of doxycycline in the media and can be combined with various molecular assays, thus providing mechanistic insights into gene function in a variety of cellular contexts.

Mutant analysis by rescue gene excision: New tools for mosaic studies in Drosophila.

A host of classical and molecular genetic tools make Drosophila a tremendous model for the dissection of gene activity. In particular, the FLP-FRT technique for mitotic recombination has greatly enhanced gene loss-of-function analysis. This technique efficiently induces formation of homozygous mutant clones in tissues of heterozygous organisms. However, the dependence of the FLP-FRT method on cell division, and other constraints, also impose limits on its effectiveness. We describe here the generation and testing of tools for Mutant Analysis by Rescue Gene Excision (MARGE), an approach whereby mutant cells are formed by loss of a rescue transgene in a homozygous mutant organism. Rescue-transgene loss can be induced in any tissue or cell-type and at any time during development or in the adult using available heat-shock-induced or tissue-specific flippases, or combinations of UAS-FLP with Gal4 and Gal80ts reagents. The simultaneous loss of a constitutive fluorescence marker (GFP or RFP) identifies the mutant cells. We demonstrate the efficacy of the MARGE technique by flip-out (clonal and disc-wide) of a Ubi-GFP-carrying construct in imaginal discs, and by inducing a known yki mutant phenotype in the Drosophila ovary.

A Resource for Manipulating Gene Expression and Analyzing cis-Regulatory Modules in the Drosophila CNS

Here, we describe the embryonic central nervous system expression of 5,000 GAL4 lines made using molecularly defined cis-regulatory DNA inserted into a single attP genomic location. We document and annotate the patterns in early embryos when neurogenesis is at its peak, and in older embryos where there is maximal neuronal diversity and the first neural circuits are established. We note expression in other tissues, such as the lateral body wall (muscle, sensory neurons, and trachea) and viscera. Companion papers report on the adult brain and larval imaginal discs, and the integrated data setsare available online (http://www.janelia.org/gal4-gen1). This collection of embryonically expressed GAL4 lines will be valuable for determining neuronal morphology and function. The 1,862 lines expressed in small subsets of neurons (<20/segment) will be especially valuable for characterizinginterneuronal diversity and function, because although interneurons comprise the majority of all central nervous system neurons, their gene expression profile and function remain virtually unexplored.

Avoiding DEET through Insect Gustatory Receptors

DEET is the most widely used insect repellent worldwide. In Drosophila olfactory receptor neurons (ORNs), DEET is detected through a mechanism employing the olfactory receptor, OR83b. However, it is controversial as to whether ORNs respond directly to DEET or whether DEET blocks the response to attractive odors. Here, we showed that DEET suppressed feeding behavior in Drosophila, and this effect was mediated by gustatory receptor neurons (GRNs). DEET was potent in suppressing feeding as <0.1% DEET elicited aversive behavior. Inhibition of feeding required multiple gustatory receptors (GRs) expressed in inhibitory GRNs. DEET stimulated action potentials in GRNs that respond to aversive compounds, and this response was lost in the Gr32a, Gr33a, and Gr66a mutants. Since 0.02% DEET elicited action potentials, we conclude that DEET directly activates of GRNs. We suggest that the effectiveness of DEET in pest control owes to its dual action in inducing avoidance simultaneously via GRNs and ORNs.

Dual Expression Lentiviral Vectors for Concurrent RNA Interference and Rescue

RNA interference (RNAi) is a powerful new tool for the selective ablation of gene expression, facilitating loss-of-function studies. However, appropriate controls are considered essential to confirm the specificity of RNAi experiments. The most stringent control is rescue of the target gene in a form that is refractory to RNAi. To facilitate rescue of the target gene, we have created improved dual expression lentiviral vectors with the ability to simultaneously drive expression of a shRNA for RNA interference and a rescue transgene in a single vector system. In proof-of-principle experiments, we ablated more than 90% of target gene expression by targeting either the open reading frame, or the 3' UTR region. Target gene expression was successfully rescued with a cDNA containing silent third-codon point mutations in the targeted region or with native cDNA when the 3' UTR was targeted. Finally, expression of the rescue transgene can be manipulated by positional cloning and appropriate promoter selection. The dual expression lentiviral vectors described here represent a versatile strategy for confirming the integrity of RNAi experiments and may facilitate functional analyses even in the absence of an established gain-of-function model system.

QS311. Dual Expression Lentiviral Vectors for Concurrent RNA Interference and Rescue: Regulation of Rescue Transgene Expression by Alteration of Cloning Site and Positional Effect

QS311. Dual Expression Lentiviral Vectors for Concurrent RNA Interference and Rescue: Regulation of Rescue Transgene Expression by Alteration of Cloning Site and Positional Effect

The role of cis-regulatory elements in the design of gene therapy vectors for inherited blindness

Background: Hereditary retinal disease is currently known to involve nearly 200 different genetic loci. There has been remarkable recent progress in the treatment of retinal disease via gene therapy in animal models using virus-based vectors. The majority of retinal diseases affect one of several cell types. In order to target expression of a rescue transgene specifically to the cells in need of therapy, it is necessary to employ a cis-regulatory element (CRE) to drive expression of the transgene specifically in those cells. Objective/methods: This review discusses the repertoire of CREs currently available for use in gene therapy vectors for treatment of retinal disease and outlines the issues that must be taken into consideration in the development of novel CREs for the purpose of gene therapy in the retina. Conclusion: There have been a number of important recent advances in the identification and characterization of retinal CREs and their utilization in gene therapy vectors. Nevertheless, future efforts to rationally manipulate existing CREs and design novel synthetic CREs for therapeutic purposes will require a better understanding of the cis-regulatory rules that govern CRE activity in vivo.

The Evolution of Gene Regulation Underlies a Morphological Difference between Two Drosophila Sister Species

Understanding the mechanisms underlying the morphological divergence of species is one of the central goals of evolutionary biology. Here, we analyze the genetic and molecular bases of the divergence of body pigmentation patterns between Drosophila yakuba and its sister species Drosophila santomea. We found that loss of pigmentation in D. santomea involved the selective loss of expression of the tan and yellow pigmentation genes. We demonstrate that tan gene expression was eliminated through the mutational inactivation of one specific tan cis-regulatory element (CRE) whereas the Tan protein sequence remained unchanged. Surprisingly, we identify three independent loss-of-function alleles of the tan CRE in the young D. santomea lineage. We submit that there is sufficient empirical evidence to support the general prediction that functional evolutionary changes at pleiotropic loci will most often involve mutations in their discrete, modular cis-regulatory elements.

Partial rescue of MeCP2 deficiency by postnatal activation of MeCP2

In humans, mutations in the X-linked MECP2 gene, are the cause of Rett syndrome (RTT), a neurodevelopmental disorder that affects mainly girls. MeCP2 binds to methylated CpGs and is thought to act as a transcriptional repressor. In male mice, deletion or targeted mutation of Mecp2 leads to lethality and causes a neuronal phenotype. Selective mutation of Mecp2 in postnatal neurons results in a similar, although delayed, phenotype, suggesting that the symptoms are caused by MeCP2 deficiency in postmitotic neurons. In agreement with this idea, expression of a Mecp2 transgene in postmitotic neurons of Mecp2-null mutant mice resulted in the phenotypical rescue of the symptoms. To assess whether postnatal activation of MeCP2 in mutant animals could also affect the progression of the disorder, we constructed a conditionally active Mecp2 "rescue transgene" that was activated between P0 and P30. The Mecp2 transgene was under the control of the CAGGS promoter and was activated by using brain specific Cre-mediated recombination. Our results indicate that postnatal, neuron-specific activation of MeCP2 as late as 2-4 weeks of age significantly prolonged the lifespan of mutant animals and delayed the onset of neurologic symptoms.

Dependence on the Lazaro Phosphatidic Acid Phosphatase for the Maximum Light Response

The Drosophila phototransduction cascade serves as a paradigm for characterizing the regulation of sensory signaling and TRP channels in vivo . Activation of these channels requires phospholipase C (PLC) and may depend on subsequent production of diacylglycerol (DAG) and downstream metabolites . DAG could potentially be produced through a second pathway involving the combined activities of a phospholipase D (PLD) and a phosphatidic acid (PA) phosphatase (PAP). However, a role for a PAP in the regulation of TRP channels has not been described. Here, we report the identification of a PAP, referred to as Lazaro (Laza). Mutations in laza caused a reduction in the light response and faster termination kinetics. Loss of laza suppressed the severity of the phenotype caused by mutation of the DAG kinase, RDGA , indicating that Laza functions in opposition to RDGA. We also showed that the retinal degeneration resulting from overexpression of the PLD was suppressed by elimination of Laza. These data demonstrate a requirement for a PLD/PAP-dependent pathway for achieving the maximal light response. The genetic interactions with both rdgA and Pld indicate that Laza functions in the convergence of both PLC- and PLD-coupled signaling in vivo.

Analysis of an even-skipped rescue transgene reveals both composite and discrete neuronal and early blastoderm enhancers, and multi-stripe positioning by gap gene repressor gradients.

The entire functional even-skipped locus of Drosophila melanogaster is contained within a 16 kilobase region. As a transgene, this region is capable of rescuing even-skipped mutant flies to fertile adulthood. Detailed analysis of the 7.7 kb of regulatory DNA 3' of the transcription unit revealed ten novel, independently regulated patterns. Most of these patterns are driven by non-overlapping regulatory elements, including ones for syncytial blastoderm stage stripes 1 and 5, while a single element specifies both stripes 4 and 6. Expression analysis in gap gene mutants showed that stripe 5 is restricted anteriorly by Krüppel and posteriorly by giant, the same repressors that regulate stripe 2. Consistent with the coregulation of stripes 4 and 6 by a single cis-element, both the anterior border of stripe 4 and the posterior border of stripe 6 are set by zygotic hunchback, and the region between the two stripes is 'carved out' by knirps. Thus the boundaries of stripes 4 and 6 are set through negative regulation by the same gap gene domains that regulate stripes 3 and 7 (Small, S., Blair, A. and Levine, M. (1996) Dev. Biol. 175, 314-24), but at different concentrations. The 3' region also contains a single element for neurogenic expression in ganglion mother cells 4-2a and 1-1a, and neurons derived from them (RP2, a/pCC), suggesting common regulators in these lineages. In contrast, separable elements were found for expression in EL neurons, U/CQ neurons and the mesoderm. The even-skipped 3' untranslated region is required to maintain late stage protein expression in RP2 and a/pCC neurons, and appears to affect protein levels rather than mRNA levels. Additionally, a strong pairing-sensitive repression element was localized to the 3' end of the locus, but was not found to contribute to efficient functional rescue.

The Enzymatic Activity ofDrosophilaAWD/NDP Kinase Is Necessary but Not Sufficient for Its Biological Function

TheDrosophila abnormal wing discs(awd) gene encodes the subunit of a protein that has nucleoside diphosphate kinase (NDP kinase) activity. Null mutations of theawdgene cause lethality after puparium formation. Larvae homozygous for such mutations have small imaginal discs, lymph glands, and brain lobes. Neither the imaginal discs nor the ovaries from such null mutant larvae are capable of further growth or normal differentiation when transplanted into suitable host larvae. This null mutant phenotype can be entirely rescued by one copy of a transgene that has 750 bp ofawdupstream regulatory DNA fused to a full-lengthawdcDNA. Tissue-specific expression of AWD protein from this rescue transgene is identical to tissue-specific expression of β-galactosidase from a reporter transgene that has the same regulatory region fused to the bacteriallac Zgene. However, this rescue transgene or reporter transgene expression pattern is only a subset of the endogenous pattern of expression detected by eitherin situhybridization or immunohistochemistry. This suggests thatawdis normally expressed in some tissues where it is not required. The null mutant phenotype cannot be rescued at all by a transgene that has 750 bp ofawdupstream regulatory DNA fused to a full-lengthawdcDNA with a mutation that eliminates NDP kinase activity by replacement of the active site histidine with alanine. This suggests that the enzymatic activity of the AWD protein is necessary for its biological function. The human genesnm23-H1andnm23-H2encode NDP kinase A and B subunits, respectively. The protein subunit encoded by either humannm23gene is 78% identical to that encoded by theDrosophila awdgene. Transgenes that have the 750-bpawdupstream regulatory DNA fused to humannm23-H2cDNA but not tonm23-H1cDNA can rescue the imaginal disc phenotype and the zygotic lethality caused by homozygosis for anawdnull mutation as efficiently as anawdtransgene. However, rescue of female sterility requires twice as muchnm23-H2expression asawdexpression. This implies that the enzymatic activity of the AWD protein is not sufficient for its biological function. The biological function may require nonconserved residues of the AWD protein that allow it to interact with other proteins.