Brain transcriptome study through CRISPR/Cas9 mediated mouse Dip2c gene knock-out
| Autor: | Hsu Htoo, Yaowu Zheng, Rajiv Kumar Sah, May Zun Zaw Myint, Noor Bahadar, Yang Chen, Farooq Hayel, Xuechao Feng, Mi Kaythi Chan, Zin Mar Oo, Salah Adlat, Fatoumata Binta Bah, Luqing Zhang |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Male Biology 03 medical and health sciences Exon Gene Knockout Techniques Mice 0302 clinical medicine Gene expression Genetics CRISPR Animals Clustered Regularly Interspaced Short Palindromic Repeats KEGG Neuropeptide signaling pathway Gene Gene knockout Gene Editing Mice Knockout Cas9 Intracellular Signaling Peptides and Proteins Brain General Medicine Neoplasm Proteins Mice Inbred C57BL 030104 developmental biology Gene Expression Regulation Mice Inbred DBA 030220 oncology & carcinogenesis Female CRISPR-Cas Systems Transcriptome Gene Deletion RNA Guide Kinetoplastida |
| Zdroj: | Gene. 758 |
| ISSN: | 1879-0038 |
| Popis: | Dip2C is highly expressed in brain and many other tissues but its biological functions are still not clear. Genes regulated by Dip2C in brain have never been studied. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems, adaptive immune systems of bacteria and archaea, have been recently developed and broadly used in genome editing. Here, we describe targeted gene deletions of Dip2c gene in mice via CRISPR/Cas9 system and study of brain transcriptome under Dip2C regulation. The CRISPR/Cas9 system effectively generated targeted deletions of Dip2c by pronuclei injection of plasmids that express Cas9 protein and two sgRNAs. We achieved targeted large fragment deletion with efficiencies at 14.3% (1/7), 66.7% (2/3) and 20% (1/5) respectively in 3 independent experiments, averaging 26.7%. The large deletion DNA segments are 160.4 kb (Dip2CΔ160kb), spanning from end of exon 4 to mid of exon 38. A mouse with two base pair deletion was generated from a single sgRNA targeting in exon 4 (Dip2cΔ2bp) by non-homologous end joining (NHEJ). Loss of gene expression for Dip2c mRNA was confirmed by quantitative real-time PCR (qPCR). Dip2C-regulated genes and pathways in brain were investigated through RNAseq of Dip2cΔ2bp. In total, 838 genes were found differentially regulated, with 252 up and 586 down. Gene ontology (GO) analysis indicated that DEGs in brain are enriched in neurological functions including ‘memory’, ‘neuropeptide signaling pathway’, and ‘response to amphetamine’ while KEGG analysis shows that ‘neuroactive ligand-receptor interaction pathway’ is the most significantly enriched. DEGs Grid2ip, Grin2a, Grin2c, Grm4, Gabbr2, Gabra5, Gabre, Gabrq, Gabra6 and Gabrr2 are among the highly regulated genes by Dip2C. Results confirm Dip2C may play important roles in brain development and function. |
| Databáze: | OpenAIRE |
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