CRISPR-LRS for mapping transgenes in the mouse genome

Microinjected transgenes, including bacterial artificial chromosomes (BACs), insert randomly in the mouse genome. Traditional methods of mapping a transgene are challenging, thus complicating breeding strategies and the accurate interpretation of phenotypes, particularly when a transgene disrupts critical coding or noncoding sequences. Here, we introduce CRISPR-Cas9 long-read sequencing (CRISPR-LRS) to ascertain transgene integration locus and estimated copy number. This method revealed integration loci for both a BAC and Cre-driver line, and estimated the copy numbers for two other BAC mouse lines. CRISPR-LRS offers an easy approach to establish robust breeding practices and accurate phenotyping of most any transgenic mouse line.

Haemocytes are critical for Drosophila melanogaster post-embryonic development, independent of control of the microbiota

Proven roles for haemocytes (blood cells) have expanded beyond the control of infections in Drosophila. Despite this, the critical role of haemocytes in post-embryonic development has long been thought to be limited to control of microorganisms during metamorphosis. This has previously been shown by rescue of adult development in haemocyte-ablation models under germ-free conditions. Here we show that haemocytes have a critical role in post-embryonic development beyond their ability to control the microbiota. Using a newly generated, strong haemocyte-specific driver line for the GAL4/UAS system, we show that specific ablation of haemocytes is pupal lethal, even under axenic conditions. Genetic rescue experiments prove that this is a haemocyte-specific phenomena. RNA-seq data suggests that dysregulation of the midgut is a critical consequence of haemocyte ablation. We believe this novel role of haemocytes during metamorphosis is a major finding for the field. This is an exciting new Drosophila model to study the precise mechanisms in which haemocytes regulate tissue development, findings from which could have far reaching implications beyond invertebrate biology.

Masculinized Drosophila females adapt their fighting strategies to their opponent

Many animal species show aggression to gain mating partners and to protect territories and other resources from competitors. Both male and female fruit flies of the species Drosophila melanogaster exhibit aggression in same-sex pairings, but the strategies used are sexually dimorphic. We have begun to explore the biological basis for the differing aggression strategies, and the cues promoting one form of aggression over the other. Here, we describe a line of genetically masculinized females that switch between male and female aggression patterns based on the sexual identity of their opponents. When these masculinized females are paired with more aggressive opponents, they increase the amount of male-like aggression they use, but do not alter the level of female aggression. This suggests that male aggression may be more highly responsive to behavioral cues than female aggression. Although the masculinized females of this line show opponent-dependent changes in aggression and courtship behavior, locomotor activity and sleep are unaffected. Thus, the driver line used may specifically masculinize neurons involved in social behavior. A discussion of possible different roles of male and female aggression in fruit flies is included here. These results can serve as precursors to future experiments aimed at elucidating the circuitry and triggering cues underlying sexually dimorphic aggressive behavior.

Transcriptome analysis for the development of cell-type specific labeling to study olfactory circuits

In each sensory system of the brain, mechanisms exist to extract distinct features from stimuli to generate a variety of behavioural repertoires. These often correspond to different cell types at some stage in sensory processing. In the mammalian olfactory system, complex information processing starts in the olfactory bulb, whose output is conveyed by mitral and tufted cells (MCs and TCs). Despite many differences between them, and despite the crucial position they occupy in the information hierarchy, little is known how these two types of projection neurons differ at the mRNA level. Here, we sought to identify genes that are differentially expressed between MCs and TCs, with an ultimate goal to generate a cell-type specific Cre-driver line, starting from a transcriptome analysis using a large and publicly available single-cell RNA-seq dataset (Zeisel et al., 2018). Despite many genes showing differential expressions, we identified only a few that were abundantly and consistently expressed only in MCs. After further validating these putative markers using in-situ hybridization, two genes, namely Pkib and Lbdh2, remained as promising candidates. Using CRISPR/Cas9-mediated gene editing, we generated Cre-driver lines and analysed the resulting recombination patterns. This analysis indicated that our new inducible Cre-driver line, Lbhd2-CreERT2, can be used to genetically label MCs in a tamoxifen dose-dependent manner, as assessed by soma locations, projection patterns and sensory-evoked responses. Hence this line is a promising tool for future investigations of cell-type specific contributions to olfactory processing and demonstrates the power of publicly accessible data in accelerating science.

Improvement on the genetic engineering of an invasive agricultural pest insect, the cherry vinegar fly, Drosophila suzukii

Background The invasive fly Drosophila suzukii has become an established fruit pest in Europe, the USA, and South America with no effective and safe pest management. Genetic engineering enables the development of transgene-based novel genetic control strategies against insect pests and disease vectors. This, however, requires the establishment of reliable germline transformation techniques. Previous studies have shown that D. suzukii is amenable to transgenesis using the transposon-based vectors piggyBac and Minos, site-specific recombination (lox/Cre), and CRISPR/Cas9 genome editing. Results We experienced differences in the usability of piggyBac-based germline transformation in different strains of D. suzukii: we obtained no transgenic lines in a US strain, a single rare transgenic line in an Italian strain, but observed a reliable transformation rate of 2.5 to 11% in a strain from the French Alps. This difference in efficiency was confirmed by comparative examination of these three strains. In addition, we used an attP landing site line to successfully established φC31-integrase-mediated plasmid integration at a rate of 10% and generated landing site lines with two attP sequences to effectively perform φC31-Recombinase Mediated Cassette Exchange (φC31-RMCE) with 11% efficiency. Moreover, we isolated and used the endogenous regulatory regions of Ds nanos to express φC31 integrase maternally to generate self-docking lines for φC31-RMCE. Besides, we isolated the promoter/enhancer of Ds serendipity α to drive the heterologous tetracycline-controlled transactivator (tTA) during early embryonic development and generated a testes-specific tTA driver line using the endogenous beta-2-tubulin (β2t) promoter/enhancer. Conclusion Our results provide evidence that the D. suzukii strain AM derived from the French Alps is more suitable for piggyBac germline transformation than other strains. We demonstrated the feasibility of using φC31-RMCE in the cherry vinegar fly and generated a set of lines that can be used for highly efficient integration of larger constructs. The φC31-based integration will facilitate modification and stabilization of previously generated transgenic lines that carry at least one attP site in the transgene construction. An early embryo-specific and a spermatogenesis-specific driver line were generated for future use of the binary expression system tet-off to engineer tissue- and stage-specific effector gene expression for genetic pest control strategies.

Identification of genes involved in the differentiation of R7y and R7p photoreceptor cells in Drosophila

The R7 and R8 photoreceptor cells of the Drosophila compound eye mediate color vision. Throughout the majority of the eye, these cells occur in two principal types of ommatidia. Approximately 35% of ommatidia are of the pale type and express Rh3 in R7 cells and Rh5 in R8 cells. The remaining 65% are of the yellow type and express Rh4 in R7 cells and Rh6 in R8 cells. The specification of an R8 cell in a pale or yellow ommatidium depends on the fate of the adjacent R7 cell. However, pale and yellow R7 cells are specified by a stochastic process that requires the genes spineless, tango and klumpfuss. To identify additional genes involved in this process we performed a genetic screen using a collection of 480 P{EP} transposon insertion strains. We identified genes that when inactivated and/or ectopically expressed in R7 cells resulted in a significantly altered percentage of Rh3 expressing R7 cells (Rh3%) from wild-type. 53 strains resulted in altered Rh3% in the heterozygous inactivation arm of the screen. 36 strains resulted in altered Rh3% in the ectopic expression arm of the screen, where the P{EP} insertion strains were crossed to a sevEP-GAL4 driver line. 4 strains showed differential effects between the two screens. Analyses of these results suggest that R7 cell fate specification is sensitive to perturbations in transcription, growth inhibition, glycoprotein ligand binding, WNT signaling, ubiquitin protease activity and Ser/Thr kinase activity, among other diverse signaling and cell biological processes.

Development of a functional genetic tool for Anopheles gambiae oenocyte characterisation: application to cuticular hydrocarbon synthesis

Oenocytes are an insect cell type having diverse physiological functions ranging from cuticular hydrocarbon (CHC) production to insecticide detoxification that may impact their capacity to transmit pathogens. To develop functional genetic tools to study Anopheles gambiae oenocytes, we have trapped an oenocyte enhancer to create a transgenic mosquito Gal4 driver line that mediates tissue-specific expression. After crossing with UAS-reporter lines, An. gambiae oenocytes are fluorescently tagged through all life stages and demonstrate clearly the two characteristic oenocyte cell-types arising during development. The driver was then used to characterise the function of two oenocyte expressed An. gambiae cyp4g genes through tissue-specific expression of UAS-RNAi constructs. Silencing of cyp4g16 or cyp4g17 caused lethality in pupae of differing timing and penetrance. Surviving cyp4g16 knockdown adults showed increased sensitivity to desiccation. Total cuticular hydrocarbon levels were reduced by approximately 80% or 50% in both single gene knockdowns when assayed in young pupa or surviving adults respectively, indicating both genes are required for complete CHC production in An. gambiae and demonstrate synergistic activity in young pupae. Comparative CHC profiles were very similar for the two knockdowns, indicating overlapping substrate specificities of the two enzymes. Differences were observed for example with reduced abundance of shorter chain CHCs in CYP4G16 knockdowns, and reduction in longer, branched chained CHCs in CYP4G17 knockdown adults. This is the first time that two cyp4gs have both been shown to be required for complete CHC production in an insect. Moreover, the generation of tagged cells and identification of an enhancer region can expediate oenocyte specific transcriptomics. The novel driver line can also be used to explore oenocyte roles in pheromone production, mating behaviour and longevity in the malaria mosquito.

Dynamics control in pipe cold rolling mill main drive

Elimination of unstable and critical states of deformed systems of pipes pilger cold rolling mill, heavy-loaded equipment of which functions under conditionsof increase dynamic loads, is mainly intended for mechanic system dynamics stabilization until the accepted level of loading. Active mechanicties in the initial dynamic model revealed, which stipulate the demonstration of pronounced parametric processes in the studied system of pipes cold rolling mill main driver. Analysis of reasons of parametric oscillations originating carried out for systems of pipes cold rolling mill main driver. Zones of dynamic instability of mechanic system functioning according to Eins–Strett diagram determined, that enable to make the choice of passive optimal rolling regimes at the stage of technological processes designing of pipes pilger cold rolling.A system of active control by main driver of pipes cold rolling mill elaborated based on its mathematical model. A structure of optimal control by angular oscillations driver line elements of pipes cold rolling mill selected and parameters ofactive control impacts of the automated servo control system determined. A mechanism of optimal control by dynamic state ofdriver line elements of pipes cold rolling mill proposed. Also proposed an outline of critical and instablestates elimination for the main driver line driver of pipes cold rolling mill, which is realized by means of adaptive active automated servo control facility. The modernized main driver line of pipes cold rolling mill is equipped by facilities and control systems, enabling to switch the initial mechanic system from a critical state into a zone ofdesired states in a servo regime. Sensors of threshold moments level of elasticity forcesare installed on the drive shaft of main driver of pipes cold rolling mill. During the drive shaft angular elastic oscillations, comprising of actual and desired parameters of mechanicsystem is made based on sensors signals. Next, a correspondent control impact is formed based on algorithm embedded into the electric drive servo control system. When the displayed point of the drive shaft reaches the desired area of system dynamic stability, the active drive control is switched off. If further the disturbing load, applied from the side of deformation seat, makes the drive line dynamic characteristics off the desired status, then the dynamic system control process is repeated in anautomated mode. Reliability of the results received confirmed by calculation example and experiments at the main driver of pipes cold rolling mill ХПТ-32.

Conditional Deletion of FOXL2 and SMAD4 in Gonadotropes of Adult Mice Causes Isolated FSH Deficiency

The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common α gonadotropin subunit and the hormone-specific FSHβ subunit (a product of the Fshb gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that Fshb expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.