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Knockout of Sheep Mstn Gene with Zinc-Finger Nuclease

Author: ZhangCunFang
Tutor: ZhangZhiYing
School: Northwest University of Science and Technology
Course: Animal Genetic Breeding and Reproduction
Keywords: Sheep MSTN ZFN Gene targeting Knock out
CLC: S826
Type: PhD thesis
Year: 2012
Downloads: 24
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Abstract

Gene targeting is a heritable genome modification of introducing an exogenously DNA fragment to repalce of an endogenous DNA segment by homologous recombination in living cells. Nonetheless, many applications of gene targeting are hindered by its inherently low frequency(<10-6) and the indispensible selection in culture to obtain positive clones. It has been observed that introduction of double strand breaks (DSBs) within the genome increases gene targeting efficiencies. Several chimeric nucleases have been developed to introuduce DSBs and increase gene targeting frequencies, especially zinc-finger nucleases (ZFNs) technology, which was widespread applied and shown high performance. ZFNs can increase the efficiency of gene targeting significantly by introducing a DSB at a unique site within a genome. Myostatin (MSTN) is a negative regulator of skeletal muscle development; its inactivation will result in increasing cell number, or generating hyperplastic muscle, larger and heavier myofibers. At present, MSTN has become the important object of study to increase animal’s production and treat the human muscle wasting. In this study, we use ZFNs to introduce a DSB at a unique site of sheep MSTN gene, and induce mutagenesis by non-homologous end-joining (NHEJ), thus inactivate the MSTN of sheep.1. The potential targeting sites that can be recognizted by ZFN were identified by online software ZiFit3.2. And one of the potential sites located in the3rd exon was selected as the targeting site. The ZFPs pools that can specifically combined with the left and right half targeting sites were constructed via the oligomerized pool engineering (OPEN) method, based on bacterial two hybrid system.2. In order to screen the positive ZFNs that can recognize and cut the special site, pAC-Ga14-VP16-ZFBS, a unique yeast reporter vector containing the targeting site, and corresponding yeast ZFN expression vectors, pLeu-LZFN and pTrp-RZFN, were constructed. Then, cotransformation of the reporter plasmid and ZFN expression plasmids intoyeast AH109was peroformed.Positive yeast candidates that can grow on the synthetic medium lacking hsitidine and adenine. And highly active ZFNs were isolated based on PCR to detect the repair event of Gal4gene. The results showed that about80%of the targeting site in yeast has been mutated, but in the negative control group, some homoglous recombination phenomenon has been discovered which showed the activities of ZFNs should be determined futher.3Two pairs of ZFPs that can specially bind with their targeting sites located in1st exon of MSTN were assembled with context dependent assembly-synthetic CoDA method and corresponding ZFN adenoviral expression vectors were constructed. Then sheep fetal fibroblast cells were infected with the recombinant adenovirus containing the ZFN expression cassette, and detection of ZFN expression by western blotting. The results demonstrated that the ZFN expression cassette contained in the adenovirus expressed normally in the sheep fetal fibroblast cells. Considering the low activity of the selected ZFN, a red fluorescent reporter vector pRS, in which the open reading frame of Ds-Red was interrupted by targeting sites, was constructed.48h post cotransfection of the sheep fetal fibroblast cells with pRS and corresponding ZFN expression plasmids, the red fluorescence can be detected with inverted fluorescent microscope, which suggested the Ds-Red was recovered. We isolated the cells with red fluorescenceby flow-cytometry and extracted the genome from the isolated cells, but no targeted mutation was detected in the cell genome.4. We used the CoDA method to synthesize two pairs of ZFNs, which included a variant of the FokI cleavage domain, ZF-Sharkey, to target the sheep MSTN gene. We tested the activity of these ZFNs using a pRGS dual-fluorescence reporter system in HEK293T cells. According to the eGFP expression level, we obtained a pair of ZFNs that can recognize and cut the MSTN targetsite in the reporter vector. Meanwhile ZF-Sharkeys cleaved DNA with higher activity than wild-type ZFNs. Finally, the ZF-Sharkeys and reporter vector were cotransfected into sheep fetal fibroblasts and two mutant cell lines were identified by flow cytometry and sequencing.Statistical analysis showed that ZFNs mediated mutation rate in cell sorting was2.5%, the mutation rate was0.0425%in the total cells.5. We also created a suicidal system, in which a suicidal nuclease expression vector harboring a pair of ZFNs expression cassette flanked by its target site such that self-induced cleavage terminates ZFN expression. The suicidal ZFN expression cassette was inserted into an eGFP reporter gene, resulting in disruption ofeGFP gene open reading frame. Subsequently, self-cleavage by the ZFN allows homologous recombination to restore the eGFP gene. eGFP expression is a marker of detection of the cells with modified genomes. Although the activity of ZFNs in cells transfected with this suicidal ZFN expression coupled with a surrogate reporter decreased only about4.5%as compared with cells transfection with conventional ZFNs expression plasmids, ZFN-associated toxicity was reduced by about40.0%. We concluded that this new suicidal ZFN expression and surrogate reporter system represents a useful improvement for genomic editing by reducing toxicity as well as allowing easy detection of edited cells by eGFP analysis.

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