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

BHPR research: qualitative1. Complex reasoning determines patients' perception of outcome following foot surgery in rheumatoid arhtritis

Background: Foot surgery is common in patients with RA but research into surgical outcomes is limited and conceptually flawed as current outcome measures lack face validity: to date no one has asked patients what is important to them. This study aimed to determine which factors are important to patients when evaluating the success of foot surgery in RA Methods: Semi structured interviews of RA patients who had undergone foot surgery were conducted and transcribed verbatim. Thematic analysis of interviews was conducted to explore issues that were important to patients. Results: 11 RA patients (9 ♂, mean age 59, dis dur = 22yrs, mean of 3 yrs post op) with mixed experiences of foot surgery were interviewed. Patients interpreted outcome in respect to a multitude of factors, frequently positive change in one aspect contrasted with negative opinions about another. Overall, four major themes emerged. Function: Functional ability & participation in valued activities were very important to patients. Walking ability was a key concern but patients interpreted levels of activity in light of other aspects of their disease, reflecting on change in functional ability more than overall level. Positive feelings of improved mobility were often moderated by negative self perception ("I mean, I still walk like a waddling duck”). Appearance: Appearance was important to almost all patients but perhaps the most complex theme of all. Physical appearance, foot shape, and footwear were closely interlinked, yet patients saw these as distinct separate concepts. Patients need to legitimize these feelings was clear and they frequently entered into a defensive repertoire ("it's not cosmetic surgery; it's something that's more important than that, you know?”). Clinician opinion: Surgeons' post operative evaluation of the procedure was very influential. The impact of this appraisal continued to affect patients' lasting impression irrespective of how the outcome compared to their initial goals ("when he'd done it ... he said that hasn't worked as good as he'd wanted to ... but the pain has gone”). Pain: Whilst pain was important to almost all patients, it appeared to be less important than the other themes. Pain was predominately raised when it influenced other themes, such as function; many still felt the need to legitimize their foot pain in order for health professionals to take it seriously ("in the end I went to my GP because it had happened a few times and I went to an orthopaedic surgeon who was quite dismissive of it, it was like what are you complaining about”). Conclusions: Patients interpret the outcome of foot surgery using a multitude of interrelated factors, particularly functional ability, appearance and surgeons' appraisal of the procedure. While pain was often noted, this appeared less important than other factors in the overall outcome of the surgery. Future research into foot surgery should incorporate the complexity of how patients determine their outcome Disclosure statement: All authors have declared no conflicts of interes

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

Acknowledgements: We thank all technical personnel at the IMPC production centres for their contributions. H.E., E.A., M.G., L.L., C.M., and L.M.J.N. were supported by Ontario Genomics and Genome Canada OGI-051, OGI-090, OGI-137 and the Canada Foundation for Innovation. M-C.B. and Y.H. were supported by the Université de Strasbourg, the CNRS, the INSERM and the ‘Investissements d’avenir’ programs (ANR-10-IDEX-0002-02, ANR-10-LABX-0030-INRT and ANR-10-INBS-07 PHENOMIN). A.C., G.C., M.M., L.T., and S.W. were supported by the Medical Research Council MC_UP_1502/3 International Mouse Phenotyping Consortium—building a functional catalogue of a mammalian genome. B.D., G.D., E.R., H.W.-J., D.A., A.B., R.R.-S., and W.S. were supported by the Wellcome Trust. P.M. and H.P. were supported by European Molecular Biology Laboratory core funding. P.K. and R.S. used services of the Czech Centre for Phenogenomics supported by the Czech Academy of Sciences RVO 68378050 and project LM2018126 Czech Centre for Phenogenomics provided by Ministry of Education, Youth and Sports of the Czech Republic, LM2015040 Czech Centre for Phenogenomics by MEYS, CZ.1.05/2.1.00/19.0395 Higher quality and capacity for transgenic models by MEYS and ERDF, CZ.1.05/1.1.00/02.0109 Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec (BIOCEV) by MEYS and ERDF, CZ.02.1.01/0.0/0.0/16_013/0001789 Upgrade of the Czech Centre for Phenogenomics by MEYS and ESIF. Research reported in this publication was supported by the NIH Common Fund, the Office of The Director and the National Human Genomic Research Institute of the National Institutes of Health (U42OD011174 and UMIHG006348 supported A.C., D.L., G.C., F.D., I.L., M.M., J.S., L.T., S.W., M.D., and J.D.H.; U42OD011175 and UM1OD023221 supported M.G., L.L., C.M., L.M.J.N., B.W., J.A.W., M.R., and K.L.; U42OD011185 and UM1OD023222 supported L.G., K.P., R.B., J.K.W., and S.A.M.; UM1HG006370 supported A.-M.M. and H.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Funder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100010269The 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