Identification of a germline-expression promoter for genome editing in Bombyx mori

• 来源期刊: 中国昆虫科学（英文版）
• 关键词: Bombyx mori nanos promoter transgenic CRISPR/Cas9
• 年，卷（期）: 2019,（6）
• 页码范围: 991-999
• 页数: 9页
• 语种: 英文
• DOI: 10.1111/1744-7917.12657

摘要

Identification of stage-and tissue-specific cis-regulatory elements will enable more precise genomic editing.In previous studies of the silkworm Bombyx mori,we identified and characterized several tissue-and sex-specific cis-regulatory elements using transgenic technology,including a female-and fat body-specific promoter,vitellogenin,testis-specific promoters,Radial spoke head I (BmR1) and beta-tubulin 4 (Bmβ4).Here we report a cis-regulatory element specific for a somatic and germ cell-expressed promoter,nanos (Bmnos).We investigated activities of three truncated promoter sequences upstream of the transcriptional initiation site sequences of Bmnos in vitro (nos-0.6kb,nos-lkb and nos-2kb) and in vivo (nos-2kb).In BmN cultured cells,all three lengths drove expression of the gene encoding enhanced green fluorescence protein (EGFP),although nos-2kb had the highest fluorescence activity.In transgenic silkworms,nos-2kb drove EGFP expression at the early embryonic stage,and fluorescence was concentrated in the gonads at later embryonic stages.In addition,this cis-regulatory element was not sex differentiated.The fluorescence intensity gradually weakened following the larval developmental stage in the gonads and were broadly expressed in the whole body.The nos-2kb promoter drove the Cas9 system with efficiency comparable to that of the broad-spectrum strong IE1 promoter.These results indicate that Bmnos is an effective endogenous cis-regulatory element in the early embryo and in the gonad that can be used in applications involving the clustered,regularly interspaced,short palindromic repeats (CRISPR)/Cas9 system.