Elevated DNA damage sensitivity in malaria parasite induced by the synergistic action between PfHsp90 inhibitor and PfRad51 inhibitor

The DNA recombinase Rad51 from human malaria parasite Plasmodium falciparum has emerged as a potential drug target due to its central role in the homologous recombination (HR) mediated double strand break (DSB) repair pathway. Inhibition of the ATPase and strand exchange activity of PfRad51, by a small molecule inhibitor B02 (3-(Phenylmethyl)-2-[(1E)-2-(3-pyridinyl)ethenyl]-4(3H)-quinazolinone), renders the parasite more sensitive towards the genotoxic agents. Here, we investigated whether the inhibition of the molecular chaperone PfHsp90 potentiates the anti-malarial action of B02. We found that PfHsp90 inhibitor 17-AAG ( 17 -(Allylamino)- 17 -demethoxygeldanamycin) exhibits strong synergism with B02 in both drug sensitive (3D7) and multi-drug resistant (Dd2) P. falciparum parasites. 17-AAG causes more than 200-fold decrease in the half-maximal inhibitory concentration (IC 50 ) of B02 in 3D7 parasites. Our results provide mechanistic insights into such profound synergism between 17-AAG and B02. We report that PfHsp90 physically interacts with PfRad51 and promotes the UV irradiation-induced DNA repair activity of PfRad51 by controlling its stability. We find that 17-AAG reduces PfRad51 protein levels by accelerating proteasomal degradation. Consequently, PfHsp90 inhibition renders the parasites more susceptible to the potent DNA damaging agent methyl-methane-sulfonate (MMS) in a dose dependent manner. Thus, our study provides a rationale of targeting PfHsp90 along with the recombinase PfRad51 for controlling malaria propagation.

Massively parallel DNA target capture using Long Adapter Single Stranded Oligonucleotide (LASSO) probes assembled through a novel DNA recombinase mediated methodology

In the attempt to bridge the widening gap from DNA sequence to biological function, we developed a novel methodology to assemble Long-Adapter Single-Strand Oligonucleotide (LASSO) probe libraries that enabled the massively multiplexed capture of kilobase-sized DNA fragments for downstream long read DNA sequencing or expression. This method uses short DNA oligonucleotides (pre-LASSO probes) and a plasmid vector that supplies the backbone for the mature LASSO probe through Cre-Loxp intramolecular recombination. This strategy generates high quality LASSO probes libraries (~46% of probes). We performed NGS analysis of the post-capture PCR amplification of DNA circles obtained from the LASSO capture of 3087 E.coli ORFs spanning from 400- to 4,000 bp. The median enrichment of all targeted ORFs versus untargeted ORFs was 30 times. For ORFs up to 1kb in size, targeted ORFs were enriched up to a median of 260-fold. Here, we show that LASSO probes obtained in this manner, are able to capture full-length open reading frames from total human cDNA. Furthermore, we show that the LASSO capture specificity and sensitivity is sufficient for target capture from total human genomic DNA template. This technology can be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning of human sequences.

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.

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.

Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae .

Lox’d in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments

The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.

193 ROBUST PROPAGATION OF SELF-RENEWING PORCINE NEURAL STEM CELLS ISOLATED FROM TRANSGENIC PIGS WITH A GFAP-CreERT2 SYSTEM CAPABLE OF CONTROLLING THE EXPRESSION OF EGFP GENES

Ample evidence has demonstrated the important roles of pigs because their anatomical, immunologic, and physiological characteristics are fairly similar to humans. In particular, their gyrencephalic brain are more comparable to humans than rodents with similar grey and white matter composition and size. In this study, we isolated and propagated the neural stem cells (GFAP-CreERT2-NSCs) from the transgenic piglet with expression of CreERT2, a fusion protein of the DNA recombinase Cre and mutated ligand-binding domain of the human oestrogen receptor, under the control of the GFAP promoter. The primary culture from tissue of porcine CreERT2 brain led to floating spherical masses of cells that revealed similar morphology and size distribution to neurospheres reported by previous studies. Quantitative analysis indicated a yield of 2.50 ± 0.44 primary spheres per 1,000 viable cells from the neocortex, versus 12.92 ± 1.67 primary spheres per 1,000 viable cells from the periventricular region (PVR) including subventricular zone. Secondary spheres (6.67 ± 1.10 spheres from neocortex versus 23.08 ± 1.96 spheres from PVR cells) were formed from primary spheres at 10 days after passage. Tertiary spheres (8.42 ± 0.99 spheres from neocortex versus 23.08 ± 1.91 spheres from PVR cells) could also be obtained after a second passage, indicating that they were proliferating in vitro. The CreERT2-NSCs showed normal 36+XY karyotype and representative NSC markers, such as NESTIN, SOX2, and VIMENTIN. After differentiation, we were able to obtain populations of astrocytes and neurons expressing GFAP and TUJ1, respectively. In summary, we verified and propagated the isolated GFAP promoter-driven CreERT2-NSCs, which would be considered a promising source of cells for treatment of central nervous system diseases.