tTARGIT AAVs mediate the sensitive and flexible manipulation of intersectional neuronal populations in mice

While Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) adeno-associated viruses (AAVs). tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) ‘Driver’ AAV and a tetracycline response element-driven, Cre-dependent ‘Payload’ AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.

Reporter gene assay for membrane fusion of extracellular vesicles

Extracellular vesicles (EVs) secreted by living cells are expected to deliver biological cargo molecules, including RNA and proteins, to the cytoplasm of recipient cells. There is an increasing need to understand the mechanism of intercellular cargo delivery by EVs. However, the lack of a feasible bioassay has hampered our understanding of the biological processes of EV uptake, membrane fusion, and cargo delivery to recipient cells. Here, we describe a reporter gene assay that can measure the membrane fusion efficiency of EVs during cargo delivery to recipient cells. When EVs containing tetracycline transactivator (tTA)-fused tetraspanins are internalized by recipient cells and fuse with cell membranes, the tTA domain is exposed to the cytoplasm and cleaved by protease to induce tetracycline responsive element (TRE)-mediated reporter gene expression in recipient cells. This assay (designated as EV-mediated tetraspanin-tTA delivery assay, ETTD assay), enabled us to assess the cytoplasmic cargo delivery efficiency of EVs in recipient cells. With the help of a vesicular stomatitis virus-derived membrane fusion protein, the ETTD assay could detect significant enhancement of cargo delivery efficiency of EVs. Furthermore, the ETTD assay could evaluate the effect of potential cargo delivery enhancers/inhibitors. Thus, the ETTD assay may contribute to a better understanding of the underlying mechanism of the cytoplasmic cargo delivery by EVs.

tTARGIT AAVs: A sensitive and flexible method to manipulate intersectional neuronal populations

SummaryWhile Cre-dependent viral systems permit the manipulation of many neuron types, some cell populations cannot be targeted by a single DNA recombinase. Although the combined use of Flp and Cre recombinases can overcome this limitation, insufficient recombinase activity can reduce the efficacy of existing Cre+Flp-dependent viral systems. We developed a sensitive dual recombinase-activated viral approach: tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) AAVs. tTARGIT AAVs utilize a Flp-dependent tetracycline transactivator (tTA) “Driver” AAV and a tetracycline response element (TRE)-driven, Cre-dependent “Payload” AAV to express the transgene of interest. We employed this system in Slc17a6FlpO;LeprCre mice to manipulate LepRb neurons of the ventromedial hypothalamus (VMH; LepRbVMH neurons) while omitting neighboring LepRb populations. We defined the circuitry of LepRbVMH neurons and roles for these cells in the control of food intake and energy expenditure. Thus, the tTARGIT system mediates robust recombinase-sensitive transgene expression, permitting the precise manipulation of previously intractable neural populations.

Using Moderate Transgene Expression to Improve the Genetic Sexing System of the Australian Sheep Blow Fly Lucilia cuprina

The sterile insect technique (SIT) is a promising strategy to control the Australian sheep blow fly Lucilia cuprina, a major pest of sheep. We have previously developed a transgenic embryonic sexing system (TESS) for this pest to facilitate the potential SIT application. TESS carry two transgenes, a tetracycline transactivator (tTA) driver and a tTA-activated pro-apoptotic effector. TESS females die at the embryonic stage unless tetracycline is supplied in the diet. However, undesired female sterility was observed in some TESS strains without tetracycline due to expression of tTA in ovaries. Here we investigate if TESS that combine transgenes with relatively low/moderate expression/activity improves the fertility of TESS females. tTA driver lines were evaluated for tTA expression by quantitative real time PCR and/or by crossing with a tTA-activated RFPex effector line. Fertility and lethality tests showed that a TESS strain containing a driver line with moderate tTA expression and an effector line showing moderate pro-apoptotic activity could recover the fertility of parental females and eliminated all female offspring at the embryonic stage. Consequently, such a strain could be further evaluated for an SIT program for L. cuprina, and such a “moderate strategy” could be considered for the TESS development in other pest species.

Starburst amacrine cells amplify optogenetic visual restoration through gap junctions

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restore vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. It is not easy to obtain efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established retinal ganglion cell (RGC)- and amacrine cell gene induction in a murine model with high efficiency using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restoration effect, we expressed the channel rhodopsin gene into RGCs and amacrine cells using this system. Then, enhancement of the visual restoration effect was observed by gene transfer not only to RGCs but also to starburst amacrine cells. It was suggested that photoresponse from amacrine cells enhanced the maintained response of ganglion cells and furthered the visual restoration effect.

Genetic Variation and Potential for Resistance Development to the tTA Overexpression Lethal System in Insects

Release of insect pests carrying the dominant lethal tetracycline transactivator (tTA) overexpression system has been proposed as a means for population suppression. High levels of the tTA transcription factor are thought to be toxic due to either transcriptional squelching or interference with protein ubiquitination. Here we utilized the Drosophila melanogaster Genetic Reference Panel (DGRP) to examine the influence of genetic variation on the efficacy of a female-specific tTA overexpression system. The level of female lethality between DGRP lines varied from 11 to 97% with a broad sense heritability of 0.89. A genome-wide association analysis identified 192 allelic variants associated with high or low lethality (P < 10−5), although none were significant when corrected for multiple testing. 151 of the variants fell within 108 genes that were associated with several biological processes including transcription and protein ubiquitination. In four lines with high female lethality, tTA RNA levels were similar or higher than in the parental tTA overexpression strain. In two lines with low lethality, tTA levels were about two fold lower than in the parental strain. However, in two other lines with low lethality, tTA levels were similar or approximately 30% lower. RNAseq analysis identified genes that were up or downregulated in the four low female lethal lines compared to the four high lethal lines. For example, genes associated with RNA processing and rRNA maturation were significantly upregulated in low lethal lines. Our data suggest that standing genetic variation in an insect population could provide multiple mechanisms for resistance to the tTA overexpression system.

Factors other than hTau overexpression that contribute to tauopathy-like phenotype in rTg4510 mice

The tauopathy-like phenotype observed in the rTg4510 mouse line, in which human tauP301L expression specifically within the forebrain can be temporally controlled, has largely been attributed to high overexpression of mutant human tau in the forebrain region. Unexpectedly, we found that in a different mouse line with a targeted-insertion of the same transgene driven by the same tetracycline-TransActivator (tTA) allele, but with even higher overexpression of tauP301L than rTg4510, atrophy and tau histopathology are delayed, and a different behavioral profile is observed. This suggests that it is not overexpression of mutant human tau alone that contributes to the phenotype in rTg4510 mice. Furthermore we show that the tauopathy-like phenotype seen in rTg4510 requires a ~70-copy tau-transgene insertion in a 244kb deletion in Fgf14, a ~7-copy tTA-transgene insertion in a 508kb deletion that disrupts another five genes, in addition to high transgene overexpression. We propose that these additional effects need to be accounted for in any studies using rTg4510, and that Tg-INDEL mutations and their impacts on phenotype should be defined for all transgenic models used in biomedical research.

A piggyBac-based toolkit for inducible genome editing in mammalian cells

We describe the development and application of a novel series of vectors that facilitate CRISPR-Cas9-mediated genome editing in mammalian cells, which we call CRISPR-Bac. CRISPR-Bac leverages the piggyBac transposon to randomly insert CRISPR-Cas9 components into mammalian genomes. In CRISPR-Bac, a single piggyBac cargo vector containing a doxycycline-inducible Cas9 or catalytically-dead Cas9 (dCas9) variant and a gene conferring resistance to Hygromycin B is co-transfected with a plasmid expressing the piggyBac transposase. A second cargo vector, expressing a single-guide RNA (sgRNA) of interest, the reverse-tetracycline TransActivator (rtTA), and a gene conferring resistance to G418, is also cotransfected. Subsequent selection on Hygromycin B and G418 generates polyclonal cell populations that stably express Cas9, rtTA, and the sgRNA(s) of interest. Using Mus musculus-derived embryonic and trophoblast stem cells, we show that CRISPR-Bac can be used to knockdown proteins of interest, to create targeted genetic deletions with high efficiency, and to activate or repress transcription of protein-coding genes and an imprinted long noncoding RNA. The ratio of sgRNA-to-Cas9-to-transposase can be adjusted in transfections to alter the average number of cargo insertions into the genome. sgRNAs targeting multiple genes can be inserted in a single transfection. CRISPR-Bac is a versatile platform for genome editing that simplifies the generation of mammalian cells that stably express the CRISPR-Cas9 machinery.