ランダム点突然変異マウスの睡眠異常スクリーニングにより、睡眠時間減少を示すマウス家系Sleepyを樹立した。この表現型は、Sik3遺伝子中のスプライスドナー部位の点突然変異によってもたらされる。見出された点突然変異をES細胞での相同組換えによりノックインした系統、すなわちSik3遺伝子に対しSleepy変異を導入したノックインマウス系統(Sik3<tm1.1Iiis>)の受精卵を用いて、CRISPR/Cas9システムによりN末2nd Met直下へFLAG-HA配列を挿入した変異型SIK3のタグノックインマウス系統である。 We induced random point mutations by ethylnitrosourea (ENU) and screened for dominant sleep and wakefulness abnormalities. Through our screening, we established a mutant pedigree, which we termed Sleepy (Slp), with a markedly prolonged sleep time. Whole-exome sequencing identified a heterozygous single nucleotide substitution at the splice donor site for intron 13 of the Sik3 gene. To confirm genetically that the Sik3 splice mutation is the sole cause of the long-sleep phenotype, we introduced the Sik3 exon 13-skipping allele by using conventional knock-in in embryonic stem (ES) cells. Further examine these functions, we introduced a Flag-HA sequence into the Sik3Slp allele using CRISPR/Cas9 technology. C57BL/6N由来のES細胞を用いてSik3遺伝子へSleepy変異をノックインした系統(Sik3<tm1.1Iiis>)を作製し、その後CRISPRによりFLAG-HAタグを挿入した。系統はC57BL/6Nで維持している。生殖細胞に明らかな異常は認められないものの、個体の生育が悪く、繁殖能が低い。 true In publishing the research results obtained by use of the BIOLOGICAL RESOURCE, a citation of the following literature(s) designated by the DEPOSITOR is requested. Nature, 539, 378-383 (2016).Prior to requesting the BIOLOGICAL RESOURCE, the RECIPIENT must obtain approval from　Dr. Masashi Yanagisawa. The availability of the BIOLOGICAL RESOURCE is limited to a RECIPIENT of a not-for profit institution for a not-for-profit research. We induced random point mutations by ethylnitrosourea (ENU) and screened for dominant sleep and wakefulness abnormalities. Through our screening, we established a mutant pedigree, which we termed Sleepy (Slp), with a markedly prolonged sleep time. Whole-exome sequencing identified a heterozygous single nucleotide substitution at the splice donor site for intron 13 of the Sik3 gene. To confirm genetically that the Sik3 splice mutation is the sole cause of the long-sleep phenotype, we introduced the Sik3 exon 13-skipping allele by using conventional knock-in in embryonic stem (ES) cells. Further examine these functions, we introduced a Flag-HA sequence into the Sik3Slp allele using CRISPR/Cas9 technology. <A HREF="https://mus.brc.riken.jp/en/mouse_of_month/mar_2019_mm" target="_blank">Mouse of the Month Mar 2019</A> Mouse line harboring a Flag-tag inserted into the N terminus of mutated SIK3 found in the Sleepy (Slp) mutant. Heterozygous males and females are prone to be poor breeders. FLP/frt system C (3-6 months) C57BL/6N-Sik3<em3Iiis> C57BL/6N-Sik3<em3Iiis> ランダム点突然変異マウスの睡眠異常スクリーニングにより、睡眠時間減少を示すマウス家系Sleepyを樹立した。この表現型は、Sik3遺伝子中のスプライスドナー部位の点突然変異によってもたらされる。見出された点突然変異をES細胞での相同組換えによりノックインした系統、すなわちSik3遺伝子に対しSleepy変異を導入したノックインマウス系統(Sik3<tm1.1Iiis>)の受精卵を用いて、CRISPR/Cas9システムによりN末2nd Met直下へFLAG-HA配列を挿入した変異型SIK3のタグノックインマウス系統である。We induced random point mutations by ethylnitrosourea (ENU) and screened for dominant sleep and wakefulness abnormalities. Through our screening, we established a mutant pedigree, which we termed Sleepy (Slp), with a markedly prolonged sleep time. Whole-exome sequencing identified a heterozygous single nucleotide substitution at the splice donor site for intron 13 of the Sik3 gene. To confirm genetically that the Sik3 splice mutation is the sole cause of the long-sleep phenotype, we introduced the Sik3 exon 13-skipping allele by using conventional knock-in in embryonic stem (ES) cells. Further examine these functions, we introduced a Flag-HA sequence into the Sik3Slp allele using CRISPR/Cas9 technology.C57BL/6N由来のES細胞を用いてSik3遺伝子へSleepy変異をノックインした系統(Sik3<tm1.1Iiis>)を作製し、その後CRISPRによりFLAG-HAタグを挿入した。系統はC57BL/6Nで維持している。生殖細胞に明らかな異常は認められないものの、個体の生育が悪く、繁殖能が低い。 C（3〜6か月） Sik3<Flag,Slp> Sik3<Flag,Slp> 睡眠覚醒に関わる遺伝子Sik3の変異型SleepyアリルへFLAG-HAタグを挿入したマウス系統。覚醒時間の短縮、加齢に伴う肥満がみられる。 RBRC09912 Necessary documents for ordering:<ol><li>Approval form (<A HREF="https://mus.brc.riken.jp/ja/wp-content/uploads/form/form_6.docx">Japanese</A> / <A HREF="https://mus.brc.riken.jp/en/wp-content/uploads/form/form_d.docx">English</A>)</li><li>Order form (<A HREF="https://mus.brc.riken.jp/ja/wp-content/uploads/form/form_4.docx">Japanese</A> / <A HREF="https://mus.brc.riken.jp/en/wp-content/uploads/form/form_b.docx">English</A>)</li><li>Category I MTA: CRISPR/Cas9 genome edited bioresources (<A HREF="https://mus.brc.riken.jp/ja/wp-content/uploads/form/Broad_MTA_J.docx">Japanese</A> / <A HREF="https://mus.brc.riken.jp/en/wp-content/uploads/form/Broad_MTA_E.docx">English</A>)</li><li>Acceptance of responsibility for living modified organism (<A HREF="https://mus.brc.riken.jp/ja/wp-content/uploads/form/form_7.docx">Japanese</A> / <A HREF="https://mus.brc.riken.jp/en/wp-content/uploads/form/form_g.docx">English</A>)</li></ol> Developed by Hiromasa Funato and Masashi Yanagisawa, University of Tsukuba. C57BL/6N background. Flag-tag was inserted into RBRC09910 by the CRISPR/Cas9 technology. 条件を付加する。<br>研究成果の公表にあたって寄託者の指定する文献を引用する。Nature, 539, 378-383 (2016).<br>利用者は提供承諾書を用いて、事前に柳沢正史博士のの承諾を得る。学術機関の学術研究に限る。 pX330-U6-Chimeric_BB-CBh-hSpCas9[human U6 promoter, S. pyogenes gRNA scaffold, human U6 terminator, CMV,chicken hybrid CMV enhancer/chicken beta-actin promotor (CBh), Synthetic DNA 3xFLAG, SV40 nuclear localization signal(NLS), Streptococcus pyogenes SpCas9 (human codon-optimized), bovine GH polyA signal, AAV2 inverted terminal repeat (ITR), f1 phage f1 origin, E. coli Ampicillin resistance gene (AmpR), E. coli pUC origin], mouse Sik3 genomic DNA, Synthetic FLAG tag, Human Influenza HA tag (Hemagglutinin gene), yeast FRT (flipase recombination target) site