本文選題：CRISPR-Cas + RS-　； 參考：《生物工程學(xué)報(bào)》2017年08期

【摘要】：嘗試?yán)肅RISPR-Cas9系統(tǒng)敲除山羊基因組中β-乳球蛋白(BLG)基因,以實(shí)現(xiàn)在BLG基因座敲入人乳鐵蛋白(h LF)基因,并進(jìn)一步探討了不同濃度RAD51蛋白激活劑(RS-1)對(duì)同源重組效率的影響。首先針對(duì)山羊BLG的第一外顯子設(shè)計(jì)并構(gòu)建了sg RNA和Cas9共表達(dá)載體p Cas9-sg BLG,將該載體轉(zhuǎn)染至山羊耳成纖維細(xì)胞,利用PCR和T7EN1法驗(yàn)證了其基因組編輯活性;然后進(jìn)一步構(gòu)建了BLG基因打靶載體p BHA-h LF-NIE(包含NEO/EGFP);將該打靶載體與p Cas9-sg BLG載體共轉(zhuǎn)染至山羊耳成纖維細(xì)胞,分別用0、5、10和20μmol/L RS-1處理細(xì)胞,分析了綠色熒光蛋白的表達(dá)效率;同時(shí)用800μg/m L G418對(duì)不同濃度RS-1處理后的細(xì)胞進(jìn)行篩選,挑取EGFP陽性細(xì)胞克隆,進(jìn)一步通過PCR和測(cè)序鑒定h LF定點(diǎn)敲入的陽性細(xì)胞克隆。結(jié)果顯示:設(shè)計(jì)的sg RNA編輯山羊BLG位點(diǎn)的效率為25%-31%;報(bào)告基因的表達(dá)效率提示RS-1可以促進(jìn)基因敲入效率的提高,其效率與RS-1濃度呈正相關(guān),20μmol/L RS-1處理組的效率是對(duì)照組的3.5倍;利用G418篩選h LF敲入陽性細(xì)胞克隆后,當(dāng)RS-1濃度為0-10μmol/L時(shí),h LF敲入效率隨著RS-1濃度增加而升高,在10μmol/L時(shí)陽性克隆率最高為32.61%,然而在20μmol/L時(shí)敲入陽性克隆率下降至22.22%,且衰老細(xì)胞克隆增多。以上結(jié)果表明,利用CRISPR-Cas9系統(tǒng)可以實(shí)現(xiàn)在山羊耳成纖維細(xì)胞中敲除BLG基因和敲入h LF基因,且適宜濃度的RS-1可以顯著提升基因敲入效率,本試驗(yàn)為高效利用CRISPR-Cas9系統(tǒng)獲得基因敲入的細(xì)胞提供了參考依據(jù)。
[Abstract]:Using CRISPR-Cas9 system to knockout 尾 -lactoglobulin (BLG) gene from goat genome, the human lactoferrin (hLF) gene was knocked into the BLG locus, and the effects of different concentrations of RAD51 protein activator (RS-1) on the efficiency of homologous recombination were further investigated. Firstly, the co-expression vector of sg RNA and Cas9 was designed and constructed for the first exon of goat BLG. The vector was transfected into goat ear fibroblasts. The genomic editing activity was verified by PCR and T7EN1. Then, the BLG gene targeting vector pBHA-h LF-NIE (including neo / EGFP) was further constructed and co-transfected with pCas9-sg BLG vector into goat ear fibroblasts. The expression efficiency of green fluorescent protein was analyzed after treated with 0 渭 mol / L and 20 渭 mol / L RS-1, respectively. At the same time, 800 渭 g / mL G418 was used to screen the cells treated with different concentrations of RS-1, and the EGFP positive cell clones were selected and further identified by PCR and sequencing. The results showed that the efficiency of the designed sg RNA editing goat BLG locus was 25-31.The expression efficiency of the reporter gene suggested that RS-1 could promote the increase of gene knockin efficiency, and the efficiency was 3.5 times higher in the 20 渭 mol / L RS-1 treatment group than in the control group. When the concentration of RS-1 was 0-10 渭 mol / L, the knockout efficiency of hLF was increased with the increase of RS-1 concentration. The highest positive clone rate was 32.61 at 10 渭 mol / L, but the knockout positive clone rate decreased to 22.22 at 20 渭 mol / L, and the number of senescent cell clones increased. These results indicate that the CRISPR-Cas9 system can be used to knockout BLG gene and hLF gene in goat ear fibroblasts, and the appropriate concentration of RS-1 can significantly improve the efficiency of gene knockout. This study provides a reference for the efficient use of CRISPR-Cas9 system to obtain knockout cells.
【作者單位】： 南京農(nóng)業(yè)大學(xué)江蘇省家畜胚胎工程實(shí)驗(yàn)室;
【基金】：國(guó)家轉(zhuǎn)基因新品種培育重大專項(xiàng)(No.2014ZX08008-004)資助~~
【分類號(hào)】：Q78

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