Pol II-expressed shRNA knocks down Sod2 gene expression and causes phenotypes of the gene knockout in mice

Abstract

RNA interference (RNAi) has been used increasingly for reverse genetics in invertebrates and mammalian cells, and has the potential to become an alternative to gene knockout technology in mammals. Thus far, only RNA polymerase III (Pol III)-expressed short hairpin RNA (shRNA) has been used to make shRNA-expressing transgenic mice. However, widespread knockdown and induction of phenotypes of gene knockout in postnatal mice have not been demonstrated. Previous studies have shown that Pol II synthesizes micro RNAs (miRNAs)-the endogenous shRNAs that carry out gene silencing function. To achieve efficient gene knockdown in mammals and to generate phenotypes of gene knockout, we designed a construct in which a Pol II (ubiquitin C) promoter drove the expression of an shRNA with a structure that mimics human miRNA miR-30a. Two transgenic lines showed widespread and sustained shRNA expression, and efficient knockdown of the target gene Sod2. These mice were viable but with phenotypes of SOD2 deficiency. Bigenic heterozygous mice generated by crossing these two lines showed nearly undetectable target gene expression and phenotypes consistent with the target gene knockout, including slow growth, fatty liver, dilated cardiomyopathy, and premature death. This approach opens the door of RNAi to a wide array of well-established Pol II transgenic strategies and offers a technically simpler, cheaper, and quicker alternative to gene knockout by homologous recombination for reverse genetics in mice and other mammalian species.

Reduced mitochondrial SOD displays mortality characteristics reminiscent of natural aging

“…Severe reduction of SOD2 expression in mice and flies causes a number of phenotypes including short life span, neuronal loss, and mitochondrial dysfunction (Li et al, 1995;Lebovitz et al, 1996;Kirby et al, 2002;Duttaroy et al, 2003;Xia et al, 2006). Here, we used Drosophila to investigate the role of this enzyme in demographic mortality parameters, nervous system pathology and behavioral senescence.…”

Identification of Loop Nucleotide Polymorphisms Affecting MicroRNA Processing and Function

“…1B and 1C) at the same site in the loop region had almost no effect on mature let-7 production, indicating that the loop sequence specificity is important for pri/pre-miRNA processing. Recently, a human miR-30-based shRNA design showed promising efficiency in gene knockdown in several cell culture and mammal systems (Silva et al, 2005;Xia et al, 2006). Our observations strongly suggested that the efficiency and specificity of miRNAbased shRNAs could be further improved by optimizing the primiR-30 loop sequence.…”

Cloning of intronic sequence within DsRed2 increased the number of cells expressing red fluorescent protein

“…3 ). Intron cloned HcRed1 construct was successfully utilized to generate transgenic miRNA-expressing cell lines and animals [4][5] and geneknockout mice for human disease research 6 . The authors show successful splicing of pre-mRNA and further excision into small miRNA, which in turn are able to trigger targeted gene silencing making this type of construct a good platform for miRNA expression 3 .…”