基于ΦC31整合酶和载体质粒pUASTattB的12株果蝇转基因阴性对照品系的建立

Generation of 12 Drosophila Transgenic Negative Control Lines Based on Site-specificΦC31 Integrase and pUASTattB Vector

目的构建基于ΦC31整合酶和载体质粒pUASTattB的果蝇转基因系统的阴性对照品系,为转基因果蝇研究实验提供更科学的阴性对照。方法用显微注射法将载体质粒pUASTattB(可携带目的基因完成定点插入的常用载体质粒)转入携带ΦC31整合酶的4种不同遗传背景的果蝇品系attP-25C6、attP-68A4、attP-75B1和attP-86F8胚胎中,培养获得G0代成虫后将每只G0代成虫分别与平衡子果蝇品系ywR13S做单管杂交(每管中G0代成虫1只与ywR13S3只进行杂交),通过观察G1代果蝇的复眼颜色,判断是否有mini-White插入,计算成功插入的概率。再选取成功插入mini-White的G1代果蝇成虫与3种平衡子果蝇品系DB、ywR13S和yw122分别做单管杂交(1只G1代雄果蝇与3只平衡子品系处女蝇杂交),平衡保种。提取保种好的果蝇品系基因组DNA,用PCR法鉴定载体质粒pUASTattB转入情况。结果4种不同遗传背景的果蝇成功显微转入pUASTattB质粒后,再用3种平衡子果蝇品系进行平衡保种,得到12株果蝇品系,均为携带mini-White标记的红眼果蝇,PCR鉴定表明有pUASTattB序列插入。结论12株转基因果蝇品系可基本满足以pUASTattB为载体构建的转基因果蝇研究实验的阴性对照需求,丰富了国家果蝇资源中心的果蝇资源。

Objective Construction of a negative control line for the Drosophilatransgenic system ORCID:0009-0000-6761-3993 based onΦC31 integrase and vector plasmid pUASTattB to provide a more scientific negative control for transgenic Drosophilaresearch experiments.Methods The vector plasmid pUASTattB was microinjected into four different genetic backgrounds Drosophila lines attP-25C6,attP-68A4,attP-75B1 and attP-86F8 embryos carryingΦC31 integrase.All of the injected embryos were incubuated to get G0 adults,and each of them was crossed with balancer stock ywR13S separately in a single vial(1 adult of the G0 generation and 3 of the ywR13S in each vial).The probability of successful insertion was calculated by observing the colour of the compound eyes of the G1 generation of Drosophila to determine whether there was a mini-White insertion.The G1 generation Drosophilaadults successfully inserted into mini-White were then selected to make single-vial crosses(one G1 generation male Drosophila crossed with three virgins of balancer Drosophila line)with each of the three balancer Drosophila strains DB,ywR13S and yw122,respectively,for balanced seed preservation.The genomic DNA of the conserved Drosophila lines was extracted and the vector plasmid pUASTattB was identified for transfer by PCR.Results 12 Drosophila strains were obtained,all of which were red-eyedDrosophilamelanogastercarrying the mini-White marker,and were identified by PCR as having the pUASTattB sequence insertion.Conclusion The 12 transgenic Drosophilastrains can meet the negative control requirements for the transgenic fly research experiments that constructed with pUASTattB as the vector basically,enriching the Drosophila resources in the National DrosophilaResource Center of China.