Efficient gene activation system on mammalian cell chromosomes using recombinant adenovirus producing Cre recombinase

CRISPR activation system is part of the function of CRSPR/Cas9 which is used to manipulate certain genes by activating these genes. CRISPR activation utilizes dCas9 (dead-Cas9) or Cas9 which is deactivated as DNA scissors, so that the use of Cas9 for gene activation does not cause targeted DNA chain termination. This research that uses this CRISPR activation is applied to the zebrafish gene (Danio rerio) aims to determine the mRNA level of the targeted gene. In this study, the ASCL1a and BCL6a genes from zebrafish were targeted as the object of study. The results showed that genes from zebrafish that were targeted had an increase in mRNA levels after being activated using CRISPR activation system. Keywords: CRISPR activation system (CRISPRa), zebrafish (Danio rerio), dCas9, ASCL1a gene, BCL6a gene

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High-Efficiency System for the Construction of Adenovirus Vectors and Its Application to the Generation of Representative Adenovirus-Based cDNA Expression Libraries

Journal of Virology ◽

10.1128/jvi.00218-06 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5435-5450 ◽

Cited By ~ 8

Author(s):

Moritz Hillgenberg ◽

Christian Hofmann ◽

Herbert Stadler ◽

Peter Löser

Keyword(s):

Human Liver ◽

Recombinant Adenovirus ◽

Adenovirus Vector ◽

Cre Recombinase ◽

Cdna Expression Library ◽

Packaging Signal ◽

Expression Library ◽

Cdna Expression ◽

Recombinant Adenovirus Vector ◽

Adenovirus Vectors

ABSTRACT We here describe a convenient system for the production of recombinant adenovirus vectors and its use for the construction of a representative adenovirus-based cDNA expression library. The system is based on direct site-specific insertion of transgene cassettes into a replicating donor virus. The transgene is inserted into a donor plasmid containing the viral 5′ inverted terminal repeat, the complete viral packaging signal, and a single loxP site. The plasmid is then transfected into a Cre recombinase-expressing packaging cell line that has been infected with a donor virus containing a partially deleted packaging signal flanked by loxP sites. Cre recombinase, by two steps of action, sequentially catalyzes the generation of a nonpackageable donor virus acceptor substrate and the generation of the desired recombinant adenovirus vector. Due to its growth impairment, residual donor virus can efficiently be counterselected during amplification of the recombinant adenovirus vector. By using this adenovirus construction system, a plasmid-based human liver cDNA library was converted by a single step into an adenovirus-based cDNA expression library with about 106 independent adenovirus clones. The high-titer purified library was shown to contain about 44% of full-length cDNAs with an average insert size of 1.3 kb. cDNAs of a gene expressed at a high level (human α1-antitrypsin) and a gene expressed at a relatively low level (human coagulation factor IX) in human liver were isolated from the adenovirus-based library using an enzyme-linked immunosorbent assay-based screening procedure.

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