Clonal analysis of gene loss of function and tissue-specific gene deletion in zebrafish via CRISPR/Cas9 technology

2016 ◽  
pp. 171-188 ◽  
Author(s):  
F. De Santis ◽  
V. Di Donato ◽  
F. Del Bene
Keyword(s):  
Gene Deletion ◽  
Gene Loss ◽  
Clonal Analysis ◽  
Specific Gene ◽  
Loss Of Function ◽  
Tissue Specific ◽  
Tissue Specific Gene

scholarly journals Highly efficient CRISPR/Cas9-mediated tissue specific mutagenesis in Drosophila

2018 ◽  
Author(s):  
Amy R. Poe ◽  
Bei Wang ◽  
Maria L. Sapar ◽  
Hui Ji ◽  
Kailyn Li ◽  
...  
Keyword(s):  
Gene Function ◽  
Gene Knockout ◽  
Somatic Cells ◽  
Specific Gene ◽  
Loss Of Function ◽  
Neuronal Morphogenesis ◽  
Tissue Specific ◽  
Guide Rna ◽  
Tissue Specific Gene ◽  
Highly Efficient

ABSTRACTTissue-specific loss-of-function (LOF) analysis is an essential approach for characterizing gene function. Here we describe an efficient CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) method for ablating gene function in Drosophila. This binary system consists of a tissue-specific Cas9 and a ubiquitously expressed multi-guide RNA (gRNA) transgene. To facilitate the construction of these components, we developed convenient tools for generating and evaluating enhancer-driven Cas9 lines, identified a multi-gRNA design that is highly efficient in mutagenizing somatic cells, and established an assay for testing the efficiency of multi-gRNAs in creating double-stranded breaks. We found that excision of genomic DNA induced by two gRNAs is infrequent in somatic cells, while indels more reliably cause tissue-specific LOF. Furthermore, we show that enhancer-driven Cas9 is less cytotoxic yet results in more complete gene removal than Gal4-driven Cas9 in larval neurons. Finally, we demonstrate that CRISPR-TRiM efficiently unmasks redundant gene functions in neuronal morphogenesis. Importantly, two Cas9 transgenes that turn on with different timings in the neuronal lineage revealed the extent to which gene products persist in cells after tissue-specific gene knockout. These CRISRPR tools can be applied to analyze tissue-specific gene function in many biological processes.

scholarly journals A unique histone 3 lysine 14 chromatin signature underlies tissue-specific gene regulation

2021 ◽  
Author(s):  
Isabel Regadas ◽  
Olle Dahlberg ◽  
Roshan Vaid ◽  
Oanh Ho ◽  
Sergey Belikov ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Specific Gene ◽  
Chromatin Signature ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Histone 3

Histones: genetic diversity and tissue-specific gene expression

1997 ◽  
Vol 107 (1) ◽  
pp. 1-10 ◽  
Author(s):  
D. Doenecke ◽  
W. Albig ◽  
C. Bode ◽  
B. Drabent ◽  
K. Franke ◽  
...  
Keyword(s):  
Gene Expression ◽  
Genetic Diversity ◽  
Specific Gene ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Specific Gene Expression

scholarly journals Blood—Brain Barrier Genomics

2001 ◽  
Vol 21 (1) ◽  
pp. 61-68 ◽  
Author(s):  
Jian Yi Li ◽  
Ruben J. Boado ◽  
William M. Pardridge
Keyword(s):  
Gene Expression ◽  
Blood Brain Barrier ◽  
Brain Barrier ◽  
Specific Gene ◽  
Microvascular Endothelium ◽  
Tissue Specific ◽  
Tissue Specific Gene ◽  
Specific Gene Expression ◽  
Blood Brain ◽  
Tester Cdna

The blood–brain barrier (BBB) is formed by the brain microvascular endothelium, and the unique transport properties of the BBB are derived from tissue-specific gene expression within this cell. The current studies developed a gene microarray approach specific for the BBB by purifying the initial mRNA from isolated rat brain capillaries to generate tester cDNA. A polymerase chain reaction–based subtraction cloning method, suppression subtractive hybridization (SSH), was used, and the BBB cDNA was subtracted with driver cDNA produced from mRNA isolated from rat liver and kidney. Screening 5% of the subtracted tester cDNA resulted in identification of 50 gene products and more than 80% of those were selectively expressed at the BBB; these included novel gene sequences not found in existing databases, ESTs, and known genes that were not known to be selectively expressed at the BBB. Genes in the latter category include tissue plasminogen activator, insulin-like growth factor-2, PC-3 gene product, myelin basic protein, regulator of G protein signaling 5, utrophin, IκB, connexin-45, the class I major histocompatibility complex, the rat homologue of the transcription factors hbrm or EZH1, and organic anion transporting polypeptide type 2. Knowledge of tissue-specific gene expression at the BBB could lead to new targets for brain drug delivery and could elucidate mechanisms of brain pathology at the microvascular level.

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