PgmNr E8005: Disruption of Sortilin-related receptor (sorl1) gene causes severe malformations, apoptosis and stunted structure in newly TALEN Knockout zebrafish Danio rerio model: Construction and molecular characterization.

Authors:
T. Saleh 1,2 ; J. Lee 1 ; S. Chang 1 ; H. Oh 1 ; K. Kim 1 ; T. Sakuma 3 ; T. Yamamoto 3 ; J. Park 1

Institutes
1) Seoul National University, Seoul, Korea; 2) Suez Canal University, Egypt; 3) Hiroshima University, Japan.

Abstract:

Tamer Said Abdelkader1,2, Ji-Min Lee1, Seo-Na Chang1, Hanseul Oh1, Kyung-Sul Kim1,  Tetsushi Sakuma3, Takashi Yamamoto3, and Jae-Hak Park1* 

Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul, South Korea.

Department of aquatic organisms, College of Environmental Agricultural Sciences, Suez Canal University, Egypt.

Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Japan

The sorl1 gene is a neuronal apolipoprotein E receptor predominantly expressed in the central nervous system. It is genetically associated with Alzheimer’s disease (AD) as a switch in the Amyloid precursor protein (APP) processing pathway. Significant reduction in the gene expression has been found in brain tissue of AD patients. TALEN plasmids were constructed using the Platinum Gate TALEN Kit. Sets of plasmids designed to disrupt the first exon of zebrafish sorl1 gene. DSB-inducing activities of constructed TALEN plasmids were evaluated using a human cell-based reporter assay. Synthesized TALEN mRNAs were injected into cytoplasm of 1-cell stage embryos 1nl containing about 100-300 ng/ul mRNAs. Microinjected eggs were maintained in E3 medium at 28ºC for further analysis. Targeted genomic loci were amplified using primers designed to anneal approximately 150–200 base pairs upstream and downstream from the expected cut site. The test analysis showed high activity of generated TALEN plasmids than positive control ZFN. The fold change of sorl1_A TALENs with sorl1_A reporter recorded value was about 4 times. Analysis of genomic DNA extracted from TALEN microinjected embryos demonstrated three mutations in the spacer sequence; alteration of Cytosine nucleotide (C) to Guanine nucleotide (G) and addition of two thymine nucleotides (T) bases on different locations. Abnormalities like bent tail, stunted structure, cardiac edema, and apoptotic cells were found in defected individuals. We therefore conclude that disruption of first exon of sorl1 gene by TALENs mRNAs causes severe malformation suggesting after reduction of App function.

Keywords: TALEN, Sorl1, knock-out, zebrafish, Danio rerio.