Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression.

The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3' UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels

Loss of miR-29a/b1 promotes inflammation and fibrosis in acute pancreatitis

MicroRNA-29 (miR-29) is a critical regulator of fibroinflammatory processes in human diseases. In this study, we found a decrease in miR-29a in experimental and human chronic pancreatitis, leading us to investigate the regulatory role of the miR-29a/b1 cluster in acute pancreatitis (AP) utilizing a conditional miR-29a/b1–KO mouse model. miR-29a/b1-sufficient (WT) and -deficient (KO) mice were administered supramaximal caerulein to induce AP and characterized at different time points, utilizing an array of IHC and biochemical analyses for AP parameters. In caerulein-induced WT mice, miR-29a remained dramatically downregulated at injury. Despite high-inflammatory milieu, fibrosis, and parenchymal disarray in the WT mice during early AP, the pancreata fully restored during recovery. miR-29a/b1–KO mice showed significantly greater inflammation, lymphocyte infiltration, macrophage polarization, and ECM deposition, continuing until late recovery with persistent parenchymal disorganization. The increased pancreatic fibrosis was accompanied by enhanced TGFβ1 coupled with persistent αSMA+ PSC activation. Additionally, these mice exhibited higher circulating IL-6 and inflammation in lung parenchyma. Together, this collection of studies indicates that depletion of miR-29a/b1 cluster impacts the fibroinflammatory mechanisms of AP, resulting in (a) aggravated pathogenesis and (b) delayed recovery from the disease, suggesting a protective role of the molecule against AP

Capivasertib combines with docetaxel to enhance anti-tumour activity through inhibition of AKT-mediated survival mechanisms in prostate cancer

Background/objective: To explore the anti-tumour activity of combining AKT inhibition and docetaxel in PTEN protein null and WT prostate tumours. Methods: Mechanisms associated with docetaxel capivasertib treatment activity in prostate cancer were examined using a panel of in vivo tumour models and cell lines. Results: Combining docetaxel and capivasertib had increased activity in PTEN null and WT prostate tumour models in vivo. In vitro short-term docetaxel treatment caused cell cycle arrest in the majority of cells. However, a sub-population of docetaxel-persister cells did not undergo G2/M arrest but upregulated phosphorylation of PI3K/AKT pathway effectors GSK3β, p70S6K, 4E-BP1, but to a lesser extent AKT. In vivo acute docetaxel treatment induced p70S6K and 4E-BP1 phosphorylation. Treating PTEN null and WT docetaxel-persister cells with capivasertib reduced PI3K/AKT pathway activation and cell cycle progression. In vitro and in vivo it reduced proliferation and increased apoptosis or DNA damage though effects were more marked in PTEN null cells. Docetaxel-persister cells were partly reliant on GSK3β as a GSK3β inhibitor AZD2858 reversed capivasertib-induced apoptosis and DNA damage. Conclusion: Capivasertib can enhance anti-tumour effects of docetaxel by targeting residual docetaxel-persister cells, independent of PTEN status, to induce apoptosis and DNA damage in part through GSK3β.</p

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for correcting variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations

Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines

The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease

Direct measurements of meltwater runoff on the Greenland ice sheet surface

Meltwater runoff from the Greenland ice sheet surface influences surface mass balance (SMB), ice dynamics, and global sea level rise, but is estimated with climate models and thus difficult to validate. We present a way to measure ice surface runoff directly, from hourly in situ supraglacial river discharge measurements and simultaneous high-resolution satellite/drone remote sensing of upstream fluvial catchment area. A first 72-h trial for a 63.1-km2 moulin-terminating internally drained catchment (IDC) on Greenland?s midelevation (1,207?1,381 m above sea level) ablation zone is compared with melt and runoff simulations from HIRHAM5, MAR3.6, RACMO2.3, MERRA-2, and SEB climate/SMB models. Current models cannot reproduce peak discharges or timing of runoff entering moulins but are improved using synthetic unit hydrograph (SUH) theory. Retroactive SUH applications to two older field studies reproduce their findings, signifying that remotely sensed IDC area, shape, and supraglacial river length are useful for predicting delays in peak runoff delivery to moulins. Applying SUH to HIRHAM5, MAR3.6, and RACMO2.3 gridded melt products for 799 surrounding IDCs suggests their terminal moulins receive lower peak discharges, less diurnal variability, and asynchronous runoff timing relative to climate/SMB model output alone. Conversely, large IDCs produce high moulin discharges, even at high elevations where melt rates are low. During this particular field experiment, models overestimated runoff by +21 to +58%, linked to overestimated surface ablation and possible meltwater retention in bare, porous, low-density ice. Direct measurements of ice surface runoff will improve climate/SMB models, and incorporating remotely sensed IDCs will aid coupling of SMB with ice dynamics and subglacial systemspublishersversionPeer reviewe

Cell-Type Specific Expression of a Dominant Negative PKA Mutation in Mice

We employed the Cre recombinase/loxP system to create a mouse line in which PKA activity can be inhibited in any cell-type that expresses Cre recombinase. The mouse line carries a mutant Prkar1a allele encoding a glycine to aspartate substitution at position 324 in the carboxy-terminal cAMP-binding domain (site B). This mutation produces a dominant negative RIα regulatory subunit (RIαB) and leads to inhibition of PKA activity. Insertion of a loxP-flanked neomycin cassette in the intron preceding the site B mutation prevents expression of the mutant RIαB allele until Cre-mediated excision of the cassette occurs. Embryonic stem cells expressing RIαB demonstrated a reduction in PKA activity and inhibition of cAMP-responsive gene expression. Mice expressing RIαB in hepatocytes exhibited reduced PKA activity, normal fasting induced gene expression, and enhanced glucose disposal. Activation of the RIαB allele in vivo provides a novel system for the analysis of PKA function in physiology

Enriching college students through study abroad: a case of Nepal Field Experience - Part 1

With a view of providing an unsurpassed opportunity to college students, who are mostly from Louisiana, in gaining a comprehensive understanding of Global Climate Change issues, we completed the first Nepal Field Experience Pilot Study Abroad from May 21-June 8, 2019. A total of fifteen students from the University of Louisiana at Lafayette, Louisiana, USA, and one graduate student from University of Arizona, Arizona, USA, participated in the program. Students examined and documented the effects of climate change impacts on agriculture, water resources, wildlife, local communities, forest resources, and other ecological and environmental settings of the country. They identified various climate change mitigation and adaptation measures that had been implemented and noted gaps between policy measures and ground realities. Research topics selected by the students included the following: climate change impacts on wildlife, water pollution, structural geology of Nepal, changing rainfall patterns and adaptation, climate change and agricultural production, geology of Kathmandu valley, air quality of Kathmandu valley, changing hydrology of glaciated landscape, climate change and geohazards, emerging diseases and pests on agricultural crops, climate change adaptation by local communities, green infrastructure and climate-smart technologies, climate change impact on drinking water sources, the roadside geology, and emerging diseases, parasites and zoonotics. Each student completed their individual research project, synthesized the results, and presented to local stakeholders in conference organized by a nonprofit nongovernmental organization, Asta-Ja Rsearch and Development Center (Asta-Ja RDC), Kathmandu, Nepal. Findings of the study reveal that Nepal is experiencing huge impacts of climate change in multiple fronts including atmospheric conditions and snowfall, temperature rise, occurrence of droughts and flooding, changes on monsoon pattern, emerging diseases and pests on crops and livestock, and declining drinking water sources. Environmental pollution, especially the air and water pollution and waste management, was very serious affecting public health, aesthetics, and even the tourism of the country. In order to reverse environmental degradation and enhance climate change adaptation, immediate implementation of effective, comprehensive, coordinated, and well-thought-out climate change adaptation and environmental initiatives are necessary. Nepal Field Experience was a lifetime learning experience for the students

Enriching college students through study abroad: a case of Nepal Field Experience - Part 2

With a view of providing an unsurpassed opportunity to college students, who are mostly from Louisiana, in gaining a comprehensive understanding of Global Climate Change issues, we completed the first Nepal Field Experience Pilot Study Abroad from May 21-June 8, 2019. A total of fifteen students from the University of Louisiana at Lafayette, Louisiana, USA, and one graduate student from University of Arizona, Arizona, USA, participated in the program. Students examined and documented the effects of climate change impacts on agriculture, water resources, wildlife, local communities, forest resources, and other ecological and environmental settings of the country. They identified various climate change mitigation and adaptation measures that had been implemented and noted gaps between policy measures and ground realities. Research topics selected by the students included the following: climate change impacts on wildlife, water pollution, structural geology of Nepal, changing rainfall patterns and adaptation, climate change and agricultural production, geology of Kathmandu valley, air quality of Kathmandu valley, changing hydrology of glaciated landscape, climate change and geohazards, emerging diseases and pests on agricultural crops, climate change adaptation by local communities, green infrastructure and climate-smart technologies, climate change impact on drinking water sources, the roadside geology, and emerging diseases, parasites and zoonotics. Each student completed their individual research project, synthesized the results, and presented to local stakeholders in conference organized by a nonprofit nongovernmental organization, Asta-Ja Rsearch and Development Center (Asta-Ja RDC), Kathmandu, Nepal. Findings of the study reveal that Nepal is experiencing huge impacts of climate change in multiple fronts including atmospheric conditions and snowfall, temperature rise, occurrence of droughts and flooding, changes on monsoon pattern, emerging diseases and pests on crops and livestock, and declining drinking water sources. Environmental pollution, especially the air and water pollution and waste management, was very serious affecting public health, aesthetics, and even the tourism of the country. In order to reverse environmental degradation and enhance climate change adaptation, immediate implementation of effective, comprehensive, coordinated, and well-thought-out climate change adaptation and environmental initiatives are necessary. Nepal Field Experience was a lifetime learning experience for the students