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

doi:10.12300/j.issn.1674-5817.2023.100

实验动物与比较医学 ›› 2023, Vol. 43 ›› Issue (5): 541-547.DOI: 10.12300/j.issn.1674-5817.2023.100

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

徐龙梅¹, 沈如凌², 范春²()(), 吴薇¹()

^1.中国科学院分子细胞科学卓越创新中心, 上海 200031
^2.上海实验动物研究中心, 上海 201203

收稿日期:2023-07-10 修回日期:2023-08-14 出版日期:2023-10-25 发布日期:2023-11-01
通讯作者:
范春（1978—），男，高级财务管理师，主要从事实验动物管理。E-mail： fanchund@hotmail.com。ORCID: 0009-0008-8817-4430；

吴薇（1980—），女，博士，高级工程师，研究方向为果蝇基因编辑、果蝇资源库管理和果蝇发育生物学。E-mail: wuwei@sibcb.ac.cn。ORCID: 0009-0000-6761-3993
作者简介:徐龙梅（1969—），女，实验师，主要从事果蝇资源库管理研究。E-mail： longmei-xu@sibcb.ac.cn；
基金资助:
国家重点研发计划项目“果蝇和线虫发育代谢资源库的系统构建与分析”专项(2021YFA0805800)

Generation of 12 Drosophila Transgenic Negative Control Lines Based on Site-specific ΦC31 Integrase and pUASTattB Vector

Longmei XU¹, Ruling SHEN², Chun FAN²()(), Wei WU¹()

^1.Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai 200031, China
^2.Shanghai Laboratory Animal Research Center, Shanghai 201203, China

Received:2023-07-10 Revised:2023-08-14 Published:2023-10-25 Online:2023-11-01
Contact:
FAN Chun (ORCID: 0009-0008-8817-4430), E-mail: fanchund@hotmail.com;

WU Wei (ORCID: 0009-0000-6761-3993), E-mail: wuwei@sibcb.ac.cn

摘要/Abstract

摘要：

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ywR13S 3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。

关键词: 黑腹果蝇, 转基因, 阴性对照, 显微注射, 平衡子

Abstract:

Objective Construction of a negative control line for the Drosophila transgenic system based on ΦC31 integrase and vector plasmid pUASTattB to provide a more scientific negative control for transgenic Drosophila research 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 carrying ΦC31 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 Drosophila adults 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-eyedDrosophila melanogaster carrying the mini-White marker, and were identified by PCR as having the pUASTattB sequence insertion. Conclusion The 12 transgenic Drosophila strains 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 Drosophila Resource Center of China.

Key words: Drosophila melanogaster, Transgenic, Negative control, Microinjection, Balancers

中图分类号:

R-332

徐龙梅, 沈如凌, 范春, 吴薇. 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[J]. 实验动物与比较医学, 2023, 43(5): 541-547.

Longmei XU, Ruling SHEN, Chun FAN, Wei WU. Generation of 12 Drosophila Transgenic Negative Control Lines Based on Site-specific ΦC31 Integrase and pUASTattB Vector[J]. Laboratory Animal and Comparative Medicine, 2023, 43(5): 541-547.

图/表 8

图1 pUASTattB载体质粒图谱

Figure 1 Profile of pUASTattB plasmid vector

表1 背景果蝇品系和平衡子果蝇品系信息列表

Table 1 The list of fly strains for transgenics and balancers

果蝇品系序号Number of fly strains	名称 Name	attP位点所在染色体位置Chromosome of attP site	背景果蝇品系（基因型） Different genetic background strains (genotype)	用途 Application
背景果蝇
1	attP-25C6	2L	y[1] M{vas-int.Dm}ZH-2A w[*]; P{CaryP}attP40	定点插入
2	attP-68A4	3L	y[1] M{vas-int.Dm}ZH-2A w[*]; P{CaryP}attP2	定点插入
3	attP-75B1	3L	vas-phi-Zh2A-VK5	定点插入
4	attP-86F8	3R	y[1] M{vas-int.Dm}ZH-2A w[*]; M{3xP3-RFP.attP}ZH-86Fb	定点插入
平衡子果蝇
1	DB		W¹¹¹⁸; Sp/CyO; TM2/TM6B	平衡保种
2	ywR13S		yw; Sp/CyO; MKRS/TM2	平衡保种
3	yw122		yw hsFlp¹²²; Sp/CyO(y⁺); TM2/TM6B	平衡保种

表2 载体质粒pUASTattB显微注射入不同背景果蝇的结果列表

Table 2 Results of different genetic background strains with pUASTattB vector by microinjection

载体质粒 Vector	背景果蝇品系 Different genetic background strains	注射胚胎数目/个 Numer of injected embryos	出幼虫数目 /条 Numer of larvae	出幼虫概率 /% Percent of larvae	出成虫数目 /只 Number of flies	出成虫概率 /% Percent of flies	红眼果蝇数目 /管 Number of red-eyed flies	红眼果蝇概率/% Percent of red-eyed flies
pUASTattB	attP-25C6	200	80	40.00	35	43.75	10	28.57
pUASTattB	attP-68A4	200	75	37.50	32	42.67	12	37.50
pUASTattB	attP-75B1	200	82	41.00	36	43.90	11	30.56
pUASTattB	attP-86F8	200	78	39.00	33	42.31	13	39.39

图2 载体质粒pUASTattB显微插入前后果蝇的眼部表型对比注：A为未注射载体质粒pUASTattB的背景果蝇品系attP-25C6，A’为attP-25C6成功转入载体质粒pUASTattB后获得的红眼果蝇；B为未注射载体质粒pUASTattB的背景果蝇品系attP-68A4，B’为attP-68A4成功转入载体质粒pUASTattB后获得的红眼果蝇；C为未注射载体质粒pUASTattB的背景果蝇品系attP-75B1，C’为attP-75B1成功转入载体质粒pUASTattB后获得的红眼果蝇；D为未注射载体质粒pUASTattB的背景果蝇品系attP-86F8，D’为attP-86F8成功转入载体质粒pUASTattB后获得的红眼果蝇。所有图片均在显微镜下放大50倍拍摄。

Figure 2 Comparison of eyes phenotypes in Drosophila before and after microinjection of the vector plasmid pUASTattBNote：A, Drosophila stock attP-25C6, without pUASTattB; A', Red-eyed flies of attP-25C6 line with pUASTattB; B, Drosophila stock attP-68A4, without pUASTattB; B', Red-eyed flies of attP-68A4 line with pUASTattB; C, Drosophila stock attP-75B1, without pUASTattB; C', Red-eyed flies of attP-75B1 line with pUASTattB; D, Drosophila stock attP-86F8, without pUASTattB; D', Red-eyed flies of attP-86F8 line with pUASTattB. All photos were taken at a magnification of 50 times under the microscope.

图 3 成功转入pUASTattB载体质粒的红眼果蝇基因组DNA注：M为1 kb Plus DNA Ladder；条带1为pUASTattB载体质粒扩增结果；条带2为pUASTattB-25C6果蝇品系鉴定结果；条带3为pUASTattB-68A4果蝇品系鉴定结果；条带4为pUASTattB-75B1果蝇品系鉴定结果；条带5为pUASTattB-86F8果蝇品系鉴定结果。的PCR鉴定电泳图

Figure 3 Identification of positive lines via PCR analysis for pUASTattB insertionNote: Lane M, 1 kb Plus DNA Ladder; Lane 1, PCR product of pUASTattB; Lane 2, PCR product of pUASTattB-25C6; Lane 3, PCR product of pUASTattB-68A4; Lane 4, PCR product of pUASTattB-75B1; Lane 5, PCR product of pUASTattB-86F8.

图4 载体质粒pUASTattB以及其成功转入4种背景果蝇获得的红眼果蝇DNA测序结果比对图

Figure 4 Multiple sequences alignment between pUASTattB vector and red-eyed pUASTattB line with pUASTattB insertion

表3 12株转基因阴性对照果蝇品系的保种结果表

Table 3 The results of balanced 12 stocks for transgenic negative control

序号 No.	背景果蝇品系 Different genetic back ground strains	平衡子果蝇品系 Balancer strains	阴性对照品系基因型 Genotype of negative control lines
1	attP-25C6	DB	W¹¹¹⁸; pUASTattB-25C6/CyO;TM2/TM6B
2	attP-25C6	ywR13S	yw; pUASTattB-25C6/CyO;MKRS/TM2
3	attP-25C6	yw122	yw hsFlp¹²²; pUASTattB-25C6/CyO(y⁺);TM2/TM6B
4	attP-68A4	DB	W¹¹¹⁸; Sp/CyO; pUASTattB-68A4/TM6B
5	attP-68A4	ywR13S	yw; Sp/CyO; pUASTattB-68A4/TM6B
6	attP-68A4	yw122	yw hsFlp¹²²; Sp/CyO(y⁺); pUASTattB-68A4/TM6B
7	attP-75B1	DB	W¹¹¹⁸; Sp/CyO; pUASTattB-75B1/TM6B
8	attP-75B1	ywR13S	yw; Sp/CyO; pUASTattB-75B1/TM6B
9	attP-75B1	yw122	yw hsFlp¹²²; Sp/CyO(y⁺); pUASTattB-75B1/TM6B
10	attP-86F8	DB	W¹¹¹⁸; Sp/CyO; pUASTattB-86F8/TM6B
11	attP-86F8	ywR13S	yw; Sp/CyO; pUASTattB-86F8/TM6B
12	attP-86F8	yw122	yw hsFlp¹²²; Sp/CyO(y⁺); pUASTattB-86F8/TM6B

表4 本研究所用的平衡子果蝇中平衡子显性标志的基本信息

Table 4 The basic information of the balancers

序号 No.	平衡子名称 Name of balancers	显性标志 Dominant marker	对应染色体 Chromosome	表型描述 Comments of phenotype
1	Wg^Sp-1（Sp*）	Sp*	2#	腹侧板第三根刚毛由1根变为2根或2根以上
2	CyO（In(2LR)O, Cy dp^lv1 cn² pr）	Cy	2#	翅膀卷曲
3	MKRS（M（3）76A¹ kar¹ ry² Sb¹）	Sb	3#	刚毛短硬
4	TM2（In（3LR）Ubx¹³⁰）	Ubx	3#	大平衡棒
5	TM6B（In(3LR)TM6, Hu e）	Hu	3#	肩部多毛

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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-specific ΦC31 Integrase and pUASTattB Vector

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