{"id":181,"date":"2018-07-19T15:43:49","date_gmt":"2018-07-19T06:43:49","guid":{"rendered":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/?page_id=181"},"modified":"2021-01-22T19:48:43","modified_gmt":"2021-01-22T10:48:43","slug":"tutorial","status":"publish","type":"page","link":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/tutorial\/","title":{"rendered":"Tutorial"},"content":{"rendered":"\n

The CNV Database for Autism Research Tutorial<\/span><\/strong>
The CNV Database for Autism Research provides you with integrative information of the cellular model of ASD with copy number variations (CNVs). Each cellular model of ASD has a unique CNV which is implicated in ASD features. The purpose of this tutorial is to help you navigate the vectors and cellular resources for ASD and to provide you with not only experimental information, but also information about genes and CNVs.

Mode selector<\/span><\/strong>
You can choose the ASD associated CNV of interest from the spread sheets titled \u201cHuman Chromosome Ideogram Viewer\u201d or \u201cMouse Cell Models\u201d. After selecting the \u201cchromosome#\u201d button, the \u201cHuman Chromosome Ideogram Viewer\u201d lists the chromosomal location, length of CNV, conserved mouse chromosomal region, and the gene\u2019s information about each CNV. The green and red lines indicate \u201cduplication (dp\/+, or dp\/-)\u201d or \u201cdeletion (df\/+)\u201d, respectively.
Information about each CNV is also available in \u201cMouse Cell Models\u201d. Each CNV is linked to two different well-known online public genomic browsers: Ensemble Genome Browser (https:\/\/www.ensembl.org\/<\/a>), established by Wellcome Trust Sanger Institute and European Bioinformatics Institute, and UCSC Genome Browser (https:\/\/genome.ucsc.edu\/<\/a>), hosted by the University of California, Santa Cruz (UCSC). If you wish to obtain chromosomal, genomic sequence, and transcripts information, please use the websites above.

If you want to see more details of our experiments, please click \u201cDetails\u201d. On this page, you can see a density map, chromosome targeting strategy, CRISPR gRNA sequence, and validation results by several methods (e.g. Southern analysis, Sequencing, Digital Droplet PCR (ddPCR), Comparative Genomic Hybridization (CGH) array). The density map was designed to determine the core region of each CNV by the cumulative number of deleted (or duplicated) regions in ASD patients based on The Simons Foundation Autism Research Initiative (SFARI) database (https:\/\/gene.sfari.org\/database\/cnv\/<\/a>).

\u201cTarget genes\u201d were designed to help users find features on individual genes, including CNVs, regardless of genetic association with ASD. \u201cGenes score\u201d reflects the strength of genetic association with ASD determined by SFARI (https:\/\/gene.sfari.org\/database\/gene-scoring\/<\/a>). \u201cGenes scores\u201d are divided into seven categories based on evidence from human genetics: Category S (Syndromic), Category 1 (High confidence), Category 2 (Strong candidate), Category 3 (Suggestive evidence), Category 4 (Minimal evidence), Category 5 (Hypothesized but untested), and Category 6 (Evidence does not support a role). If you are interested in scoring information and\/or learning more about what certain scores represent, please visit the \u201cScoring Criteria Page\u201d in SFARI (https:\/\/gene.sfari.org\/about-gene-scoring\/criteria\/<\/a>). Note that these scores will be changed by the SFARI team based on ASD research advances. Users can also get individual gene information from several well-known online public browsers, National Center for Biotechnology Information (NCBI) (https:\/\/www.ncbi.nlm.nih.gov\/<\/a>), Mouse Genome Informatics (MGI), hosted by Jackson Laboratory (http:\/\/www.informatics.jax.org\/), International Neuroinformatics Organization Facility (INCF) Japan NODE, hosted by international non-profit neuroinformatics organization (https:\/\/www.neuroinf.jp\/<\/a>), Brain Transcriptome Database (BrainTX), hosted by RIKEN INCF (http:\/\/www.cdtdb.neuroinf.jp\/CDT\/Top.jsp<\/a>), and Virtual Brain with 3D-ISM (Vibrism), by RIKEN INCF (http:\/\/vibrism.neuroinf.jp\/<\/a>).

Quick search<\/strong><\/span>
CNVs and gene of interests can be easily found by using the site search located at the top of the site (https:\/\/www.med.kobe-u.ac.jp\/asddb\/<\/a>). Chromosome of interests can also be found on the \u201cMouse Cell Models\u201d page (https:\/\/www.med.kobe-u.ac.jp\/asddb\/ranking\/<\/a>).

Supplemental experimental information<\/strong><\/span>
Mouse embryonic stem (ES) cells culture
We use CMTI-2 mouse ES cells (#CMTI-2; Merck-Millipore) with male genotype which are derived from murine strain C57\/BL6J ES cells. They are grown on a feeder layer of mitotically inactivated mouse embryonic fibroblast (MEF) cells. 0.1% Gelatin (#G2625; SIGMA) coated dishes are used in mouse ES cells culture. MEF cells are cultured with Glasgow Minimum Essential Medium (GMEM, #G5154; SIGMA) with 10% FBS (fetal bovine serum, heatinactivated).

[Medium composition for mouse ES cells culture]
DMEM, High Glucose\u00a0500 ml (#11995-065; Gibco)
15% (v\/v) FBS (Fetal Bovine Serum, embryonic stem cell-qualified), heat inactivated
2 mM L-glutamine (100\u00d7 stock 25030-081; Gibco)
Penicillin\/Streptomycin (#26253-84; Nakalai tesque)
1xMEM non-essential amino acids (NEAA 100\u00d7 stock 11140-050; Gibco)
Nucleosides (1x)*
0.1 mM 2-mercaptoethanol (#M6250; SIGMA)
1000 U\/ml murine LIF (ESGRO #ESG1107, 10^7U\/ml; Merck-Millipore)

*100x Nucleoside mixture
Adenosine (#A-4036; Sigma)\u00a0 80 mg
Guanosine (#G-6264; Sigma)\u00a0 85 mg
Cytidine (#C-4654); Sigma)\u00a0 73 mg
Uridine (#U-6381; Sigma)\u00a0 73 mg
Thymidine (#T-1895; Sigma)\u00a0 24 mg
Add to 100 ml distilled water, and then dissolved by warming to 37C.
Filtrate and then aliquot. Store at -20C.

Vectors and Cells<\/strong><\/span>
The targeting vector cassette (empty vector), DT-A\/loxP\/PGK-Neo-pA\/loxP, is available from RIKEN, Animal Resource Development & Genetic Engineering Unit (CLST, Japan). (http:\/\/www2.clst.riken.jp\/arg\/index.html<\/a>). Vector map (http:\/\/www2.clst.riken.jp\/arg\/Cassette\/CassetteMap_09.html<\/a>) and vector sequence (http:\/\/www2.clst.riken.jp\/arg\/Cassette\/CassetteSEQ_09.html<\/a>) are also available from RIKEN CLST. For the CRISPR-Cas9 vector cassette, we used pX330 developed by Dr. Feng Zhang (Massachusetts Institute of Technology (MIT), USA). To obtain these empty vectors, you may need to contact a researcher or Addgene (https:\/\/www.addgene.org\/<\/a>. Vectors developed by our team have been donated to RIKEN BRC Gene Engineering Division http:\/\/dna.brc.riken.jp\/en\/<\/a>). Researchers can obtain these via RIKEN BRC. Targeted Cells are also available from RIKEN BRC \u201cCELL BANK\u201d (http:\/\/dna.brc.riken.jp\/en\/<\/a>). All experiments involving chromosome targeting were conducted using the Nucleofector 2b device (#AAB-1001; LONZA) with Mouse Embryonic Stem Cell Nucleofector Kit (#VAPH-1001; LONZA).The targeting vector cassette (empty vector), DT-A\/loxP\/PGK-Neo-pA\/loxP, is available from RIKEN, Animal Resource Development & Genetic Engineering Unit (CLST, Japan). (http:\/\/www2.clst.riken.jp\/arg\/index.html<\/a><\/u>). Vector map (http:\/\/www2.clst.riken.jp\/arg\/Cassette\/CassetteMap_09.html<\/a><\/u>) and vector sequence (http:\/\/www2.clst.riken.jp\/arg\/Cassette\/CassetteSEQ_09.html<\/a><\/u>) are also available from RIKEN CLST. For the CRISPR-Cas9 vector cassette, we used pX330 developed by Dr. Feng Zhang (Massachusetts Institute of Technology (MIT), USA). To obtain these empty vectors, you may need to contact a researcher or Addgene (https:\/\/www.addgene.org\/<\/a><\/u>. Vectors developed by our team have been donated to RIKEN BRC Gene Engineering Division http:\/\/dna.brc.riken.jp\/en\/<\/a><\/u>). Researchers can obtain these via RIKEN BRC. Targeted Cells are also available from RIKEN BRC \u201cCELL BANK\u201d (http:\/\/dna.brc.riken.jp\/en\/<\/a><\/u>). All experiments involving chromosome targeting were conducted using the Nucleofector 2b device (#AAB-1001; LONZA) with Mouse Embryonic Stem Cell Nucleofector Kit (#VAPH-1001; LONZA).<\/p>\n","protected":false},"excerpt":{"rendered":"

The CNV Database for Autism Research TutorialThe CNV Database for Autism Research provides you with integrative information of the cellular model of ASD with copy number variations (CNVs). Each cellular model of ASD has a unique CNV which is implicated in ASD features. The purpose of this tutorial is to help you navigate the vectors and cellular resources for ASD and to provide you with not only experimental information, but also information about genes and CNVs. Mode selectorYou can choose the ASD associated CNV of interest from the spread sheets titled \u201cHuman Chromosome Ideogram Viewer\u201d or \u201cMouse Cell Models\u201d. After selecting the \u201cchromosome#\u201d button, the \u201cHuman Chromosome Ideogram Viewer\u201d lists …<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"footnotes":""},"class_list":["post-181","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/pages\/181","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/comments?post=181"}],"version-history":[{"count":20,"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/pages\/181\/revisions"}],"predecessor-version":[{"id":237,"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/pages\/181\/revisions\/237"}],"wp:attachment":[{"href":"https:\/\/www.med.kobe-u.ac.jp\/asddb\/wp-json\/wp\/v2\/media?parent=181"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}