MOUSE MODEL A new synapse disease discovered at the base of Autism and intellectual disabilities.
April 20, 2017
A new synapse disease discovered at the base of Autism and intellectual disabilities.
An international collaborative study coordinated by the Inserm unit 930 "Imaging and Brain" (Dr. Frédéric Laumonnier, Tours), PHENOMIN and the IGBMC (Dr. Yann Hérault, Illkirch) brings new and original data on the characterization of the physiopathological role of the synaptic receptor PTCHD1. Published on April 20 in the journal Molecular Psychiatry, this work reveals that the loss of function of this gene involved in intellectual disability and autism, leads to synaptic dysfunction.
Synapses are zones of contact between neurons, ensuring the connection and propagation of the information between them, and causing their excitation or inhibition. During the development of the brain, the formation of synapses is essential for brain functions such as memory and learning.
Genetic mutations involved in autism and intellectual disability (ID) seem to cause defects in the structure and function of these synapses.
In the Research Unit "Imaging and Brain" at François-Rabelais University in Tours, Frédéric Laumonnier and his colleagues are interested in the synaptic receptor PTCHD1 (Patched Domain containing 1), for which genetic mutations are associated with an ID and / or autism in boys. After creating a pathological mouse model deficient for this gene, through the Gencodys Consortium (Genetic and Epigenetic Networks in Cognitive Dysfunction; http://www.gencodys.eu/), scientists from PHENOMIN and the IGBMC showed major memory defects, as well as significant symptoms of hyperactivity on this model. Parallel studies by the team of Frédéric Laumonnier revealed changes in the synapses. The latter discovered significant alterations in synaptic structure and activity in excitatory neuronal networks of the hippocampus, a region of the brain that plays a major role in cognitive processes.
This work defines a new synapse disease occurring during development, in technical terms a neuro-developmental synaptopathy, caused by the deficiency of the PTCHD1 gene associated with ID and autism. Understanding the pathophysiological mechanisms underlying these neurodevelopmental disorders is essential to improve therapeutic strategies.
Read the recent publication in Molecular Psychology
SUM UP 2nd Programmable Nucleases Course – SUM UP
April 10, 2017
Dear colleagues and friends,
Last week we had the 2nd edition of the Nuclease hands-on Course at the CCP-BioCev, in Prague, nicely organized by Radislav Sedlacek, Inken Beck and colleagues. I think it was a great course, where participants (and instructors) could learn a lot and discuss about their own projects directly with people actively developing or using CRISPR tools. We enjoyed the Czech hospitality and the impressive Czech Centre for Phenogenomics, an excellent new and modern venue to host this type of hands-on courses and also where to successfully run mouse functional genomics projects, from generation to phenotyping.
I would like to highlight three aspects of this course, which might be relevant or hopefully be of some interest for some of you.
1.- Francis Stewart delivered a great talk sharing the latest results from his lab in using CRISPR stategies, combined with recombineering (a technique he devised and for which he was awarded the ISTT Prize at the TT2010 meeting in Berlin), for improved knockin projects. The bottom line and his take-home message was to return to use classical targeting constructs (that can be obtained through recombineering techniques) with up to 4 kb (in total, both arms) of homology, within a plasmid, delivered as intact circular DNA (hence closed). Combining the use of these targeting consctructs, where recombineering could be most helpful for building them, with a guide RNA opening the locus where we expect the knockin to occur in mouse ES cells resulted in great efficiencies, in anycase greater than using oligo-based strategies, apparently simpler and quicker, but not necessarily more efficient, as we all know. Whether the same high knockin efficiencies using plasmid templates carrying targeting constructs can be observed or not in mouse embryos is something currently being investigated. In anycase, please keep one eye in current and forthcoming Francis papers. A couple of his recent contributions are:
RecET direct cloning and Redαβ recombineering of biosynthetic gene clusters, large operons or single genes for heterologous expression.
Wang H, Li Z, Jia R, Hou Y, Yin J, Bian X, Li A, Müller R, Stewart AF, Fu J, Zhang Y.
Nat Protoc. 2016 Jul;11(7):1175-90.
RAC-tagging: Recombineering And Cas9-assisted targeting for protein tagging and conditional analyses.
Baker O, Gupta A, Obst M, Zhang Y, Anastassiadis K, Fu J, Stewart AF.
Sci Rep. 2016 May 24;6:25529.
2.- Marie Christine Birling and Guillaume Pavlovic, from the Institute Clinique de la Souris (ICS-Phenomin-IGBMC-Univ Strasbourg), directed by Yann Herault, presented their amazing set of results using CRISPR strategies to obtain almost any type of gross chromosomal alterations, including deletions, duplications, inversions, alone or in any combination, both in rats and in mice, in their attempts to reproduce in rodents some of the features associated with Down syndrome. Some very big deletions were achived in rats (up to 24.4 Mb), setting new world records in this field. Most of these results are included in their last publication:
Efficient and rapid generation of large genomic variants in rats and mice using CRISMERE.
Birling MC, Schaeffer L, André P, Lindner L, Maréchal D, Ayadi A, Sorg T, Pavlovic G, Hérault Y.
Sci Rep. 2017 Mar 7;7:43331. doi: 10.1038/srep43331.
This study nicely illustrates the power but also the allelic variability of results one can achive using CRISPR strategies when attempting to trigger deletions, duplications and inversions. Most of the time the planned alleles will be found among the founder mice generated, and transmitted through germline to their progeny, but one has to invest time and carefully design genotyping strategies to adequately interpret the great variety of genome-edited alleles generated. This study also highlights that rats would appear better hosts for these type of challenging CRISPR-mediated genome alterations, and, in general for knockin attempts as well, considering the efficiencies reported, as compared to mice. Guillaume speculated that perhaps the fact that rats are mostly outbred whereas mice used are mostly inbred would play a role regarding what can be easily done or not done with them, at genome editing level. Something interesting to take into account and further investigate.
3.- Bernd Zetsche (BROAD-MIT), from Feng Zhang's lab, was also participating in this course and delivered a great talk on their newest data, as well as data from other labs, on the Cpf1 nuclease, the novel CRISPR-Clas II nuclease, alternative to Cas9, they discovered and first reported in 2015 (http://www.ncbi.nlm.nih.gov/pubmed/26422227). As you know, the Cpf1 has unique and interesting properties, different from Cas9. Cpf1 uses a PAM located at 5', it appears to cut leaving protruding ends and only requires one short RNA guide (not two, as Cas9). Besides acting as RNA driven DNA endonuclease has also been found to act as RNAse, being able to process a multiple array of crRNA guides from a single RNA into the respective short RNA guides later used to target DNA sequences at genomic loci. Cpf1 seems to be used a lot in the Plant world. Our colleagues from plants appear to have discovered that Cpf1 is more attractive than Cas9 in producing genome-edited modifications, perhaps due to the additional simplicity of Cpf1 (one RNA, crRNA), as compared to Cas9 (two RNAs, crRNA and tracrRNA). The combined DNA and RNA nuclease activities of Cpf1 were recently reported by Bernd and colleagues in this publication:
Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F. Nat Biotechnol. 2017 Jan;35(1):31-34.
Bernd also shared the latest news concerning the PAM specificity of Cpf1. This nuclease was originally reported to use a PAM at 5' consisting of a recognition sequence of TTN or TTTN, but this is no longer correct. Updated experiments and results have led to the conclusion that the correct PAM for Cpf1 nucleases at 5' is "TTTV", where V can be A, G or C, but not T. The matching sequence (the lenght of the RNA guide) was originally announced as 23, but the reality is that only the first 18 positions contribute to drive the specificity, the homology, whereas the last four positions, from 20 to 23, can be in fact any nucleotide "NNNN", therefore these potential off-targets are in reality, and must be considered as, on-targets! Also, Bernd announced a new set of Cpf1 mutants derived in Feng Zhang's lab, with altered PAM specificities. In particular two of these mutants are the so-called Cpf1 RVR, whose PAM is "TVTV" and the so-called Cpf1 RR, whose PAM is "TYCV". These two mutants were reported in a manuscript currently under review but already posted at the BioRchiv pre-print server. Bernd announced that these two Cpf1 mutants will be soon available through AddGene. The deposited Cpf1 wild-type plasmid has one NLS but recent data, particularly in cells, suggest that an additional NLS must be added to ensure efficient translocation into the nucleus, something which might be not that relevant when injecting these reagents into mouse embryos, particularly if injected into pronucleus. In this regard, it was noted that IDT is the first company commercializing Cpf1 protein (https://eu.idtdna.com/pages/products/genome-editing/crispr-genome-editing/crispr-cpf1-genome-editing). Disclosure: I have no interests with IDT products
Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Nat Biotechnol. 2016 Aug;34(8):863-868.http://www.ncbi.nlm.nih.gov/pubmed/27272384
Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK. Nat Biotechnol. 2016 Aug;34(8):869-874.http://www.ncbi.nlm.nih.gov/pubmed/27347757
Engineered Cpf1 Enzymes with Altered PAM Specificities. Linyi Gao, David B.T. Cox, Winston X Yan, John Manteiga, Martin Schneider, Takashi Yamano, Hiroshi Nishimasu, Osamu Nureki, Feng Zhang. BioRxiv, Dec 4, 2016. doi: https://doi.org/10.1101/091611http://biorxiv.org/content/early/2016/12/04/091611
In vivo high-throughput profiling of CRISPR-Cpf1 activity. Kim HK, Song M, Lee J, Menon AV, Jung S, Kang YM, Choi JW, Woo E, Koh HC, Nam JW, Kim H. Nat Methods. 2017 Feb;14(2):153-159.https://www.ncbi.nlm.nih.gov/pubmed/27992409
The fact that new PAM sequences are required for devising correct RNA guides to be used in combination with Cpf1 prompted me to contact our bioinformatician at CNB-CSIC, Juan Carlos Oliveros, with whom we generated and launched Breaking-Cas (https://www.ncbi.nlm.nih.gov/pubmed/27166368). He efficiently responded to my request and already implemented the changes and now, you can seek for Cpf1 cutting sites at your favourite genomic sequence using the updated version of Breaking-Cas (click on nuclease presetting and select either wild-type or mutant Cpf1). I believe Breaking-Cas is currently the only RNA guide designing web-based program where Cpf1 guides can be designed with the correct PAM sequences.http://bioinfogp.cnb.csic.es/tools/breakingcas/
All these CRISPR-related updates and improvements have been annotated in the CRISPR web page we maintain at CNB-CSIC.
with best regards
Lluis (Dr. Lluis Montoliu Investigador Cientifico - Research Scientist CSIC- Centro Nacional de Biotecnologia (CNB-CSIC)- Campus de Cantoblanco
C/ Darwin, 3 28049 Madrid (Spain) Tel. +34-91-5854844 / Fax +34-91-5854506 e-mail: email@example.com
NEW OFFER Integrated approach for bone pathology
April 10, 2017
PHENOMIN-ICS offers new capabilities for analysing bone dynamics including both in vivo and ex vivo analysis of trabecular and cortical bone structures by µCT. This augments our current capacities of skeletal examinations by TRAP histological stain, X-ray, DEXA scanning, and quantitative NMR.
Contact us for help in designing your experiments on bone dynamics!
Picture: Representative 3D volumes illustrating trabecular microarchitecture in the caudal vertebra (left) and cortical bone in the tibia (right) of Sham-operated and Ovarectomized (OVX) mice, 9 weeks after surgery.
OPEN LECTURES 2nd Programmable Nucleases Course – OPEN LECTURES
April 3, 2017
As part of the CCP programmable nuclease course, the following lectures will be open lectures.Lectures will be held in the main BIOCEV conference hall (Kinosál) Průmyslová 595, Vestec and selected lectures (Live at IMG) will be broadcasted live in the Milan Hašek Auditorium at IMG.
G. Pavlovic and MC Birling from PHENOMIN-ICS give 4 open lectures !
Live at IMG Guillaume Pavlovic and Marie-Christine Birling, PHENOMIN-ICS, France
Title: “Repertoire of gene targeting methods in rodents”
Guillaume Pavlovic, PHENOMIN-ICS, France
Title: “Expanding the Research Applications for CRISPR”
Live at IMG Marie-Christine Birling, PHENOMIN-ICS, France
Title: “Genome editing using nucleases: new possibilities and application to reveal gene function in diseases”
Live at IMG Guillaume Pavlovic,PHENOMIN-ICS, France
Title: “from genotype to phenotype”
WORKSHOP CELPHEDIA Workshop Meeting 2017
May 4, 2017
CELPHEDIA is a network including fifteen centers spread throughout the French territory to be closer to the users. This distributed operational RI (Research Infrastructure) gathers a remarkable palette of expertise, skills and knowledge unique in Europe, in the domains of creation, functional exploration, archiving and distribution of organisms, used as models for fundamental research and preclinical approaches: rodents with the mouse as a leader, non-human primates and non mammalians including aquatic vertebrates.
CELPHEDIA Network gathering work packages composed of experts in their respective field, proposes to every WP member to meet during CELPHEDIA Workshop Meeting 2017.
The aim of the workshop is to continue the development of the current collaborative cross-center projects, but also to optimise and harmonise protocols and processes in the areas specific to each WP.
The Operational Committee is divided in workpackages following 4 main topics:
The working groups correspond to the objectives of CELPHEDIA nodes and gather all the scientific experts of the network in a given area.
Due to its unique combination of expertise and skills, CELPHEDIA enables to develop transverse inter-species approaches, at the level of both the technology and the study of human diseases.
On one hand, these WPs materialize this combination of skills through exchanges between the centers. On the other hand, they enable the harmonization of protocoles and processes, either scientific or administrative, to provide users with services specific to the network through CELPHEDIA gateway.
CELPHEDIA Workshop Meeting 2017 will take place at CNRS headquarters, 3 Rue Michel-Ange in Paris.
More information at http://www.celphedia.eu/
CONGRESS 43e colloque AFSTAL 2017
June 7 - 9, 2017
Attend the French congress in animal laboratory sciences
Le 43e Colloque de notre Association, qui se tiendra du 7 au 9 juin 2017 dans la ville de Lyon et dont le titre sera “Éthologie en Expérimentation Animale”.
Dans la lignée des éditions précédentes, la première session du congrès sera consacré à la présentation des plateformes locales et des équipes scientifiques de la région lyonnaise. Puis, le colloque laissera place à l’ensemble des conférences à “l’Ethologie dans tous ses états" en portant un regard particulier sur le comportement individuel et social des animaux, les neurosciences et les sciences cognitives. L’aspect scientifique, expérimental et fonctionnel de l’éthologie sera ainsi abordé.
Cette année encore une attention toute particulière sera portée aux retours d’expérience. C’est pourquoi nous retrouverons au sein du programme de ce colloque, des communications libres et des ateliers de fournisseurs.
La plus grande exposition commerciale d’Europe sera accessible durant la totalité du colloque afin de favoriser les discussions et les échanges entre les congressistes et les exposants. En effet, tous les partenaires, fournisseurs, distributeurs de matériels et de services, incontournables dans le domaine des sciences des animaux de laboratoire, seront au rendez-vous. À cette occasion, tout en participant au programme scientifique, vous pourrez découvrir les dernières nouveautés et développer votre réseau professionnel et personnel.
Pour le Comité d’organisation
Frédéric Jacquot, Président de l’AFSTAL
More information at http://www.colloque-afstal.com/2017/index.php
SPECIAL EVENT 2d European Advanced School for Mouse Phenogenomics
June 12 - 16, 2017
Would you like to learn how to choose, create and analyse your best mouse model?
Do you have a sufficient background in mouse functional genomics to lead as best your research?
Then, come to the heart of Europe to be involved - together with other students and researchers from all over the world - in opened discussion, in constructive analysis of your own research and in building conceptual scientific advances.This unique international event will provide the attendees with an innovative program focused on the best practices in the use of the mouse model for biomedical and basic research.
You will gain crucial skills required to use as best mutant mice (best experimental design, scientific and ethic consideration for you best experimental design, awareness for improving robustness of your scientific data,..)
24 experts from European landscape will be present to:
The target audience consists of engineers and/or researchers, clinicians, who directly or indirectly use mouse as animal model for scientific research.
We encourage both industrial and academic researchers, postdocs and PhDs from all Europe to attend this school.
More information on program and registration: https://advanced-school.phenomin.eu/
Contact : firstname.lastname@example.org
More information at https://advanced-school.phenomin.eu/
TRAINING COURSE Mycoplasmes detection in cell culture
October 18, 2017
Engineers and technicians currently cultivating cell lines or planning to cultivate cell lines.
None, except understand French
The course is conducted in French
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TRAINING COURSE Small animal imaging: evolution, potential, limits
November 6 - 10, 2017
The course is conducted in French
CNRS formation entreprises
MEETING 9 ieme colloque des belles souris
November 9, 2017
Le but du Club des Belles Souris est de susciter des échanges scientifiques autour des technologies et techniques en génétique moléculaire de la souris.
Le programme scientifique du 8 ième Colloque est ouvert !
Vous pouvez vous inscrire dès maintenant en utilisant le site web
More information at http://stemcells.free.fr/?page_id=1175
TRAINING COURSE In vivo CRISPR-Cas9 genome editing
November 9 - 10, 2017
PHENOMIN's training aims to provide a general framework to get scientists started using CRISPR/Cas9 for in vivo gene editing in rodents.
The main topics are:
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