scholarly journals Moving forward with forward genetics: A summary of the INFRAFRONTIER Forward Genetics Panel Discussion

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 456
Author(s):  
Asrar Ali Khan ◽  
Michael Raess ◽  
Martin Hrabe de Angelis
Keyword(s):  
Mouse Models ◽  
Gene Function ◽  
Essential Gene ◽  
Genetic Disorders ◽  
Panel Discussion ◽  
Forward Genetics ◽  
Mouse Strains ◽  
Sequence Information ◽  
International Mammalian Genome ◽  
Shed Light

In the last few decades, forward genetics approaches have been extensively used to identify gene function. Essentially, forward genetics is the elucidation of the genetic basis of a specific phenotype by screening a population containing random genomic modifications that alter gene function. These approaches have shed light on some essential gene functions in development and disease and have expanded the realm of understanding for genetic disorders. Due to the availability of efficient mutagenesis methods, phenotyping techniques, reliable validation, comprehensive sequence information and translational potential, mouse models are favored for forward genetics approaches. However, in this post-genomic CRISPR-Cas9 era, the relevance and future of forward genetics was brought into question. With more than 7300 mouse strains archived and close interactions with several leading mouse researchers around the world, INFRAFRONTIER - the European Research Infrastructure for mouse models organised a panel discussion on forward genetics at the International Mammalian Genome Conference 2018 to discuss the future of forward genetics as well as challenges faced by researchers using this approach in the current research environment. The commentary presents an overview of this discussion.

scholarly journals Current recommendations for clinical surveillance and genetic testing in rhabdoid tumor predisposition: a report from the SIOPE Host Genome Working Group

2021 ◽  
Author(s):  
M. C. Frühwald ◽  
K. Nemes ◽  
H. Boztug ◽  
M. C. A. Cornips ◽  
D. G. Evans ◽  
...  
Keyword(s):  
Working Group ◽  
Soft Part ◽  
Genetic Disorders ◽  
Panel Discussion ◽  
Rhabdoid Tumor ◽  
Host Genome ◽  
Cancer Surveillance ◽  
Malignant Neoplasms ◽  
Pathogenic Variants ◽  
Increased Risk

AbstractThe rhabdoid tumor (RT) predisposition syndromes 1 and 2 (RTPS1 and 2) are rare genetic conditions rendering young children vulnerable to an increased risk of RT, malignant neoplasms affecting the kidney, miscellaneous soft-part tissues, the liver and the central nervous system (Atypical Teratoid Rhabdoid Tumors, ATRT). Both, RTPS1&2 are due to pathogenic variants (PV) in genes encoding constituents of the BAF chromatin remodeling complex, i.e. SMARCB1 (RTPS1) and SMARCA4 (RTPS2). In contrast to other genetic disorders related to PVs in SMARCB1 and SMARCA4 such as Coffin-Siris Syndrome, RTPS1&2 are characterized by a predominance of truncating PVs, terminating transcription thus explaining a specific cancer risk. The penetrance of RTPS1 early in life is high and associated with a poor survival. However, few unaffected carriers may be encountered. Beyond RT, the tumor spectrum may be larger than initially suspected, and cancer surveillance offered to unaffected carriers (siblings or parents) and long-term survivors of RT is still a matter of discussion. RTPS2 exposes female carriers to an ill-defined risk of small cell carcinoma of the ovaries, hypercalcemic type (SCCOHT), which may appear in prepubertal females. RT surveillance protocols for these rare families have not been established. To address unresolved issues in the care of individuals with RTPS and to propose appropriate surveillance guidelines in childhood, the SIOPe Host Genome working group invited pediatric oncologists and geneticists to contribute to an expert meeting. The current manuscript summarizes conclusions of the panel discussion, including consented statements as well as non-evidence-based proposals for validation in the future.

IMMU-22. THE IMPACT OF MICROGLIA MODULATION ON GBM PROGRESSION IN SYNGENEIC MOUSE MODELS

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi96-vi96
Author(s):  
Marie-Françoise Ritz ◽  
Tala Shekarian ◽  
Tomás A Martins ◽  
Philip Schmassmann ◽  
Gregor Hutter
Keyword(s):  
Mouse Models ◽  
Tumor Development ◽  
Adaptive Immune Response ◽  
Tamoxifen Treatment ◽  
Mouse Strains ◽  
Intracerebral Injection ◽  
Adaptive Immune ◽  
Tumor Immune Microenvironment ◽  
The Impact

Abstract BACKGROUND The tumor immune microenvironment (TME) of Glioblastoma consists of almost myeloid-derived macrophages and microglia called TAMs. We have shown that the disruption of CD47-Sirpα-axis induces an antitumor activity of TAMs against GBM in immune-deficient mice, through increases of phagocytosis of tumor cells by TAMs. We have aimed to study the role of microglia and its activation/depletion on GBM progression, in the syngeneic GBM model in immune-competent mice. We have studied the interplay of innate and adaptive immune response after activation and depletion of microglia and the effect on tumor progression and outcome of the mice. MATERIAL AND METHODS We used different colonies of genetically modified immunocompetent mouse strains to genetically activate/deplete microglia in the tumor context. We generated Sall1 CreERT2/fl mice and Cre-negative littermates. The application of Tamoxifen in this constellation leads to the excision of the transcription factor Sall1 and subsequent enhanced microglia activity. Conversely, we generated Sall1 CreERT2 x Csf1r fl/fl animals and the respective heterozygous and Cre-negative littermates in which Tamoxifen treatment leads to inactivation of microglia through the deletion of Csf1r. Glioblastoma tumors were induced by intracerebral injection of GL261, CT2A, or retrovirus-induced PDGF-Akt in pups and Tamoxifen treatment was started once the tumors were detected. RESULTS We observed a survival advantage in tumor-bearing mice after activation of microglia in Sall1 CreERT/fl animals compared to Cre-negative littermates. Genetic depletion of microglia in this model resulted in a shorter lifespan in microglia-depleted animals compared to Cre-negative littermates. Furthermore, the iTME in these tumors is subjected to scRNAseq analysis to identify mechanistic insights. CONCLUSION Microglia are important players in tumor development and progression of glioblastoma in mouse models. These cells may be targeted in future immunotherapeutic approaches for patients.

scholarly journals Generation, quality control, and analysis of the first genomically humanised knock-in mice for the ALS/FTD genes SOD1, TARDBP (TDP-43), and FUS

2021 ◽  
Author(s):  
Anny Devoy ◽  
Georgia Price ◽  
Francesca De Giorgio ◽  
Rosie Bunton-Stasyshyn ◽  
David Thompson ◽  
...  
Keyword(s):  
Quality Control ◽  
Mouse Models ◽  
Splice Variants ◽  
Region Of Interest ◽  
Human Protein ◽  
Mouse Strains ◽  
Coding Region ◽  
Protein Biochemistry ◽  
Oxford Nanopore

Amyotrophic lateral sclerosis - frontotemporal dementia spectrum disorder (ALS/FTD) is a complex neurodegenerative disease; up to 10% of cases are familial, usually arising from single dominant mutations in >30 causative genes. Transgenic mouse models that overexpress human ALS/FTD causative genes have been the preferred organism for in vivo modelling. However, while conferring human protein biochemistry, these overexpression models are not ideal for dosage-sensitive proteins such as TDP-43 or FUS. We have created three next-generation genomically humanised knock-in mouse models for ALS/FTD research, by replacing the entire mouse coding region of Sod1, Tardbp (TDP-43) and Fus, with their human orthologues to preserve human protein biochemistry, with exons and introns intact to enable future modelling of coding or non-coding mutations and variants and to preserve human splice variants. In generating these mice, we have established a new-standard of quality control: we demonstrate the utility of indirect capture for enrichment of a region of interest followed by Oxford Nanopore sequencing for robustly characterising large knock-in alleles. This approach confirmed that targeting occurred at the correct locus and to map homologous recombination events. Furthermore, extensive expression data from the three lines shows that homozygous humanised animals only express human protein, at endogenous levels. Characterisation of humanised FUS animals showed that they are phenotypically normal compared to wildtype littermates throughout their lifespan. These humanised mouse strains are critically needed for preclinical assessment of interventions, such as antisense oligonucleotides (ASOs), to modulate expression levels in patients, and will serve as templates for the addition of human ALS/FTD mutations to dissect disease pathomechanisms.

scholarly journals No audible wheezing

2005 ◽  
Vol 201 (12) ◽  
pp. 1869-1873 ◽  
Author(s):  
Joshua A. Boyce ◽  
K. Frank Austen
Keyword(s):  
Mast Cells ◽  
Mouse Models ◽  
Airway Hyperresponsiveness ◽  
Pulmonary Inflammation ◽  
Genetic Differences ◽  
Mouse Strains ◽  
T Helper ◽  
Th2 Cell ◽  
Helper Type

Mouse models of T helper type 2 (Th2) cell–biased pulmonary inflammation have elucidated mechanisms of sensitization, cell traffic, and induced airway hyperresponsiveness (AHR). Nonetheless, most mice lack intrinsic AHR, a central property of human asthma, and disparities persist regarding the contributions of eosinophils and mast cells and the sensitivity to induced AHR in the commonly used mouse strains. We suggest that these discordances, reflecting methodological and genetic differences, may be informative for understanding heterogeneity of human asthma.

scholarly journals A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michelle Spoto ◽  
Changhui Guan ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Gene Function ◽  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Model Organisms ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Wide Range ◽  
User Friendly

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

scholarly journals The discovery and importance of genomic imprinting

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Anne C Ferguson-Smith ◽  
Deborah Bourchis
Keyword(s):  
Gene Expression ◽  
Genomic Imprinting ◽  
Genetic Disorders ◽  
Epigenetic Inheritance ◽  
Specific Gene ◽  
Specific Gene Expression ◽  
Epigenetic Control ◽  
International Award ◽  
Parent Of Origin ◽  
Shed Light

The discovery of genomic imprinting by Davor Solter, Azim Surani and co-workers in the mid-1980s has provided a foundation for the study of epigenetic inheritance and the epigenetic control of gene activity and repression, especially during development. It also has shed light on a range of diseases, including both rare genetic disorders and common diseases. This article is being published to celebrate Solter and Surani receiving a 2018 Canada Gairdner International Award "for the discovery of mammalian genomic imprinting that causes parent-of-origin specific gene expression and its consequences for development and disease".

Factor IX: Insights from knock-out and genetically engineered mice

2008 ◽  
Vol 100 (10) ◽  
pp. 563-575 ◽  
Author(s):  
Paul E. Monahan
Keyword(s):  
Mouse Models ◽  
Positional Cloning ◽  
Coagulation Factors ◽  
Genetically Engineered ◽  
Factor Ix ◽  
Mouse Strains ◽  
Knock Out ◽  
Haemophilia B ◽  
Genetically Engineered Mice ◽  
Knock Out Mice

SummaryThe study of coagulation factors has been rapidly advanced by studies performed in genetically engineered mouse strains. Investigation of factor IX (FIX) has benefited from excellent genedeleted mouse models that recapitulate many of the features of human haemophilia B. Moreover, advanced positional cloning techniques and availability of technology to allow not only knock-out mice, but also knock-in and knock-down mice, provide new opportunities to observe genotype-phenotype and structure-function correlations regarding FIX, as well as the interaction of FIX with inflammatory, immune, and tissue repair systems. In this paper, available FIX knock-out mice and additional haemophilia B mouse models are reviewed specifically in regards to observations these models have facilitated concerning: factor IX gene expression and factor IX protein pharmacokinetics; the role of FIX in haemostasis, thrombosis and wound healing; insights into coagulation FIX arising out of gene therapy applications in haemophilia mouse models; immunology of tolerance or loss of tolerance of FIX and inhibitor antibody formation.

scholarly journals Investigating essential gene function inMycobacterium tuberculosisusing an efficient CRISPR interference system

2016 ◽  
Vol 44 (18) ◽  
pp. e143-e143 ◽  
Author(s):  
Atul K. Singh ◽  
Xavier Carette ◽  
Lakshmi-Prasad Potluri ◽  
Jared D. Sharp ◽  
Ranfei Xu ◽  
...  
Keyword(s):  
Gene Function ◽  
Essential Gene ◽  
Crispr Interference ◽  
Interference System

A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1416-1427 ◽  
Author(s):  
E.A. Silva ◽  
B.J. Lee ◽  
L.S. Caceres ◽  
D. Renouf ◽  
B.R. Vilay ◽  
...  
Keyword(s):  
Gene Function ◽  
Imaginal Disc ◽  
Essential Gene ◽  
Cellular Responses ◽  
Multiple Alleles ◽  
Gene Mutant ◽  
Nonessential Gene ◽  
Disc Cells ◽  
Complementation Groups ◽  
Novel Strategy

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc29DE gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc95E mutants, which are extremely sensitive to caffeine. Although huc95E is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc95E gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc29D and huc95E as a first step toward molecularly characterizing the relevant genes.

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