Disruption of Me<scp>CP</scp>2 attenuates circadian rhythm in <scp>CRISPR</scp>/Cas9‐based Rett syndrome model mouse

Tsuchiya, Yoshiki; Minami, Yoichi; Umemura, Yasuhiro; Watanabe, Hidenobu; Ono, Daisuke; Nakamura, Wataru; Takahashi, Tomoyuki; Honma, Sato; Kondoh, Gen; Matsuishi, Toyojiro; Yagita, Kazuhiro

doi:10.1111/gtc.12305

Cited by 32 publications

(38 citation statements)

References 45 publications

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“…We have shown that approximately threefold numbers of oocytes could be collected from C57BL/6 female mice at 4 weeks of age by the administration of IASe compared with the conventional administration of IAS or eCG alone. We and other investigators have confirmed that fertilized oocytes created by IVF via the conventional superovulation method were applicable for creating genome-edited mice (Nakagawa et al, 2014, 2015; Li et al, 2014; Miura et al, 2015; Tsuchiya et al, 2015; Kaneko and Mashimo, 2015; Nakao et al, 2016; Sakurai et al, 2016). However, it has not yet been elucidated whether the ultra-superovulation technique can be applied for the generation of genome-edited mice.…”

Section: Introductionmentioning

confidence: 58%

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

et al. 2016

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Current advances in producing genetically modified mice using genome-editing technologies have indicated the need for improvement of limiting factors including zygote collection for microinjection and their cryopreservation. Recently, we developed a novel superovulation technique using inhibin antiserum and equine chorionic gonadotropin to promote follicle growth. This method enabled the increased production of fertilized oocytes via in vitro fertilization compared with the conventional superovulation method. Here, we verify that the ultra-superovulation technique can be used for the efficient generation of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated knockout mice by microinjection of plasmid vector or ribonucleoprotein into zygotes. We also investigated whether single-amino-acid-substituted mice and conditional knockout mice could be generated. Founder mice bearing base substitutions were generated more efficiently by co-microinjection of Cas9 protein, a guide RNA and single-stranded oligodeoxynucleotide (ssODN) than by plasmid microinjection with ssODN. The conditional allele was successfully introduced by the one-step insertion of an ssODN designed to carry an exon flanked by two loxP sequences and homology arms using a double-cut CRISPR-Cas9 strategy. Our study presents a useful method for the CRISPR-Cas9-based generation of genetically modified mice from the viewpoints of animal welfare and work efficiency.

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Section: Introductionmentioning

confidence: 58%

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

et al. 2016

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“…However, this was not statistically significant ( P =0.6). The 18 previously published studies studies that have targeted individual loci in the mouse genome with single sgRNAs have not specifically addressed the symmetry of deletions101117183132333435363738394041424344. Deleted sequences were available from seven studies11183134394344 (Supplementary Data 1), but only one18 showed large enough data sets to permit a direct comparison with ours.…”

Section: Resultsmentioning

confidence: 99%

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

et al. 2017

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Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

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“…The C57BL/6 strain has been widely used as a genetic background for congenic and mutant mice because genome mapping has been done [4]. Recently, this strain is often used for gene manipulation using clustered, regularly interspaced short palindromic repeat/CRISPR-associated (CRISPR/Cas) and transcription activator-like effector nucleases (TALEN) [43][44][45]. For these reasons, our new FSGS model mice may be useful in renal studies using genetic manipulation and help uncover the molecular mechanisms behind podocytopathy.…”

Section: Discussionmentioning

confidence: 99%

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

Takeuchi

Naito

Kawashima

et al. 2017

Nephron

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Background: Focal segmental glomerulosclerosis (FSGS) is considered a subset of the podocytopathies. The molecular pathogenesis of podocytopathy is still unknown. There has not been an experimental animal model of isolated podocytopathy induced by antibody in C57BL/6 strain, which is widely used as the genetic background. Nephrin is closely associated with the slit diaphragm of the glomerular podocyte, and has recently received attention as a potential therapeutic target. The function of nephrin, especially its role in FSGS development via podocytopathy, is being elucidated. We report our experience with a C57BL/6 FSGS model induced by polyclonal rabbit anti-mouse nephrin antibody (α-mNep Ab). Methods: α-mNep Ab, which was generated by genetic immunization, was administered into C57BL/6 mice at once, intravenously. Urinary protein excretion, the development of glomerulosclerosis and the number of podocyte in mouse kidney were evaluated. Results: The α-mNep Ab-induced FSGS was associated with massive proteinuria and nephrotic syndrome. In periodic acid-Schiff staining, FSGS was observed from day 7 after antibody injection. Podocyte numbers and podocyte marker (anti-Wilms tumor 1 and anti-synaptopodin)-positive areas were clearly decreased. These results suggest that this FSGS mouse model reliably reproduces the human nephrotic syndrome and FSGS. Conclusion: We succeeded in making the nephrotic syndrome model mice induced by α-mNep Ab using C57BL/6. This model may be useful for studying the mechanisms of podocytopathy.

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Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9‐based Rett syndrome model mouse

Cited by 32 publications

References 45 publications

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome

New Anti-Nephrin Antibody Mediated Podocyte Injury Model Using a C57BL/6 Mouse Strain

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