利用CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入研究

为研究CRISPR/Cas9腺病毒载体在鸡胚中进行基因敲入的可行性,将包装不同滴度增强型绿色荧光报道基因(Enhance green fluorescent protein,EGFP)的腺病毒载体和慢病毒载体显微注射到HH14时期鸡胚的外周血管中,对胚胎发育至3.5 d和9 d鸡胚存活、各器官中EGFP荧光强度等指标进行检测;将包装CRISPR/Cas9系统的腺病毒载体和Donor片段的腺病毒载体共同显微注射到鸡胚后,对成年公鸡各器官中EGFP荧光长期表达、EGFP基因敲入等指标进行检测。结果显示:两种病毒载体对鸡胚心脏和性腺的转染效率呈现显著的滴度依赖性,但1×1011TU/mL的EGFP腺病毒载体将9 d鸡胚存活率从93.18%显著降低至64.11%(P<0.05);选择滴度为1×1010TU/mL的CRISPR/Cas9腺病毒和Donor腺病毒载体进行EGFP的基因敲入,注射后成年公鸡多个组织均能稳定表达绿色荧光,PCR检测证明该荧光来自EGFP的基因敲入。研究提示:腺病毒载体比慢病毒载体具有更高的鸡胚转染效率,使用CRISPR/Cas9腺病毒载体可以在鸡胚中实现高效的EGFP基因敲入。

Specific Gene Insertion in Chicken Embryos using Adenovirus Vector Carrying CRISPR System

This study was aimed to explore the feasibility of CRISPR/Cas9 adenovirus for gene insertion in chicken embryos. Different titers of adenoviral vector and lentiviral vector with enhanced green fluorescent protein(EGFP) were microinjected into the blood vessels of chicken embryos at HH14 stage. The survival rate of chicken embryos at 3.5 and9 days was calculated. The fluorescence intensity of EGFP in each organ of the 9-day chicken embryo was measured.After the adenovirus vector carrying CRISPR/Cas9 system and the donor fragment were co-microinjected into the chicken embryos, the hatching rate of eggs was detected, and the fluorescent expression of EGFP in various organs of adult roosters was detected.PCR verifies whether EGFP has been knocked in.The results showed that the transfection efficiency of viral vectors to the heart and gonads of chicken embryos was significantly titer-dependent, but the 1×1011 TU/mL EGFP adenovirus vector significantly reduced the survival rate of chicken embryos at 9 days from 93.18% to 64.11%.The CRISPR/Cas9 adenovirus with a titer of 1×1010 TU/mL was selected for EGFP gene knock-in, and the results showed that multiple tissues of adult roosters could express green fluorescence stably for a long time. PCR detection proved that the fluorescence originated from the knock-in of EGFP. The overall results suggested that adenoviral vectors have higher transfection efficiency of chicken embryos than lentiviral vectors, and using CRISPR/Cas9 adenoviral vectors can achieve efficient gene knock-in in chicken embryos.