Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

被引:22
作者
Elaswad, Ahmed [1 ,2 ]
Khalil, Karim [1 ,3 ]
Cline, David [1 ]
Page-McCaw, Patrick [4 ]
Chen, Wenbiao [4 ]
Michel, Maximilian [5 ]
Cone, Roger [5 ]
Dunham, Rex [1 ]
机构
[1] Auburn Univ, Sch Fisheries Aquaculture & Aquat Sci, Auburn, AL 36849 USA
[2] Suez Canal Univ, Fac Vet Med, Dept Anim Wealth Dev, Ismailia, Egypt
[3] Cairo Univ, Fac Vet Med, Anat & Embryol Dept, Giza, Egypt
[4] Vanderbilt Univ, Dept Mol Physiol & Biophys, Nashville, TN 37232 USA
[5] Univ Michigan, Life Sci Inst, Ann Arbor, MI 48109 USA
来源
JOVE-JOURNAL OF VISUALIZED EXPERIMENTS | 2018年 / 131期
关键词
Genetics; Issue; 131; CRISPR/Cas9; gene editing; gene knockout; microinjection; channel catfish embryos; mutation; truncated protein; indels; CARP CYPRINUS-CARPIO; CRISPR-CAS9; SYSTEM; HORMONE GENE; ZEBRAFISH; ELECTROPORATION; EFFICIENT; NUCLEASE; EXPRESSION; SALMON; DISRUPTION;
D O I
10.3791/56275
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.
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页数:12
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