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FLP/FRT Induction of Mitotic Recombination in Drosophila Germline
果蝇种系中有丝分裂重组的FLP/FRT诱导   

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Abstract

The FLP/FRT system is a site-directed recombination technology based on the targeting of a recombination enzyme (flipase - FLP) to specific DNA regions designated as flipase recognition target (FRT) sites. Initially identified in Saccharomyces cerevisiae, the yeast FLP-enzyme and its FRT recombination targets were successfully transferred into each major chromosome arm in Drosophila (Golic and Lindquist, 1989). This offers the ability to mediate mitotic recombination in vivo during development in a controlled manner [revised in Theodosiou and Xu (1998)]. The controlled induction of the mitotic recombination events is usually performed by expressing the FLP under the control of the heat-shock (hs) promoter. This allows the expression of high FLP levels at specific developmental time windows. Strains carrying these genetically marked FLP/FRT chromosomes have greatly enhanced our ability to study gene function in both germline and somatic Drosophila tissues. Here we describe two different protocols: One to induce and identify homozygous mutant clones in ovaries and the other to generate female germline mutants for the analysis of maternal effects on embryogenesis.

Keywords: Drosophila(果蝇), FLP/FRT(FLP/FRT), Mitotic Recombination(有丝分裂重组), Germline(生殖系)

Materials and Reagents

  1. Fly stocks
    1. w; FRT 42B, fand/CyO (Guilgur et al., 2014)
    2. y w hs-FLP22/Y; FRT 42B nls-GFP/CyO hs-hid (Guilgur et al., 2014)
    3. y w hs-FLP22; If/CyO hs hid (Guilgur et al., 2014)
    4. FRT 42B ovoD/T(1;2)OR64/CyO (Guilgur et al., 2014; Bloomington Drosophila Stock Center, catalog number: 4434)
  2. Molasses (Sipa Barley Malt48, Provida, catalog number: G109115B )
  3. Bett syrup (Zuckerrubensirup, catalog number: 01939 )
  4. Cornmeal (Provida)
  5. Yeast granulated (LESAFFRE IBÉRICA)
  6. Soy flour (Salutem, catalog number: 5601557003008 )
  7. Agar ( Nzytech, catalog number: MB02904 )
  8. Napagin (Dutscher, catalog number: 789063 )
  9. Propionic acid (Acros Organics)
  10. Fly food (see Recipes)

Equipment

  1. Plastic bottles
  2. Water bath at 37 °C
  3. Stereoscope

Procedure

  1. Generation and labeling of mutant clones using the FLP/FRT system
    In this section we describe the generation of homozygous mutant clones for a recessive mutation fandango (fand) which has been characterized in Guilgur et al. (2014). In order to discriminate between fand homozygous mutant and wild-type clones, we combine the FLP/FRT system with a cell-autonomous marker as originally described in Xu and Rubin (1993). We use Green Fluorescent Protein tagged with a nuclear localization signal (nls-GFP) in order to easily identify clones based on the presence (wild type clones-nls-GFP plus) or absence (mutant clones-nls-GFP minus) of a nuclear GFP signal (Figure 1).
    Flies were raised using standard techniques at 25 °C unless indicated. All crosses were set up in bottles and flipped to new food vials on a daily basis at least 4 times.
    1. To generate homozygous mosaic clones of fandango allele we crossed virgins carrying the mutant allele (genotype: w; FRT 42B, fand/CyO) with males carrying the following markers: hs-flipase, the FRT 42B, the nls-GFP construct and a balancer CyO bearing the heat-shock-inducible pro-apoptotic transgene head involution defective (hid) (genotype: y w hs-FLP22/Y; FRT 42B nls-GFP/CyO hs-hid). The reverse cross could also be used (see Figure 2).
    2. By the late second/third larval instar stages the F1 progeny was heat-shocked in a 37 °C water bath for 1 h, in order to induce mitotic recombination and the lethality of organisms carrying the balancer with hid transgene. An additional 1 h heat-shock in the following day could be done to increase the frequency of clones.
      Note: The developmental stage selected to induce the mitotic recombination via heat shock can vary depending on the tissue to be analyzed. Doing two consecutive heat shock could increase the rate of recombination as a result of more flipase expression and therefore more clones will be generated. However, it is important be aware about the health of the original stocks used in the cross (sometimes mutant stocks are weak) and more than one heat shock could be harmful for the flies. In our case for the fand allele it was sufficient one heat shock. The number of flies used in the cross referred in section 1 are approximately 15 female virgins with 5 males.


      Figure 1. Generating and labeling mutant clones using FLP/FRT system and a cell marker. In a heterozygous parental cell (A), FLP induces mitotic recombination between FRT sites (yellow arrow) on homologous 2R chromosome arms. Segregation of recombinant chromosomes at mitosis produces two daughter cells: A mutant cell bearing two copies of the mutant allele fand (D) and a wild-type cell containing only the wild-type form of the gene (C). The cell marker nls-GFP co-segregates with the wild-type gene (wild type twin-spot clones - nls-GFP plus) and the mutant clone cells are labeling by its absence (fand mutant clones - nls-GFP minus). The non-recombined cells are also identified by the lighter signal from only one copy of nls-GFP (B) (wild type one-spot clones - nls-GFP plus). Schematic representation of clone mosaics in Drosophila ovaries (E). FLP-recombinase target sequences (FRT) are depicted in cyan, fandango mutation in orange and the nls-GFP construct in ochre, heterozygous parental cell in light brown, one-spot cells in light green, twin-spot cells in green, fand mutant clone cells in pink.


      Figure 2. Genetic crosses to recover FRT/FLP induced clones labeled by a cell marker

    3. F1 adult females with normal wings (absence of curly wings indicates lack of the CyO balancer) were transferred to food vials supplemented with fresh baker’s yeast for 3 days prior to ovary dissection and processing (Figure 3A-B).


      Figure 3. Fandango mutant clones in ovaries. Oogenesis is normal in fandango mutant ovary clones (A, B). Absence of endogenous nls-GFP (nls-GFP minus) indicated that the cells were homozygous for fand mutation. Contrary, presence of endogenous nls-GFP (nls-GFP plus) indicates wild type clones. Ovaries were stained for F-actin (red) and WGA (blue).

  2. Generation of fandango maternal mutant embryos from germline mutant clones using the FLP/FRT OvoD system
    In this section we describe the generation of female germline mutant clones to characterize the maternal effect of the recessive zygotic lethal mutation fandango (Guilgur et al., 2014). The genetic technique applied in this assay takes advantage of the properties of the yeast “FLP/FRT” site-specific recombination system in combination with the germline-dependent dominant female sterile OvoD mutation [originally described in Chou and Perrimon (1992)] (Figure 4).


    Figure 4. Generating germline mutant clones using FLP/FRT and OvoD system. In a heterozygous parental cell (A), FLP induces mitotic recombination between FRT sites (yellow arrow) on homologous 2R chromosome arms. Segregation of recombinant chromosomes at mitosis produces two daughter cells: a mutant germ cell bearing two copies of the mutant allele fand (D) and a wild-type cell containing only the wild-type form of the gene (C). The presence of germ line-dependent dominant female sterile OvoD mutation blocks oogenesis generating atrophic ovaries (B, C). Therefore, all the developed ovaries are homozygous mutant for fand allele. Schematic representation of the generation of germline clone ovaries during Drosophila oogenesis (E). FLP-recombinase target sequences (FRT) are depicted in cyan, fandango mutation in orange, the OvoD dominant mutation in ochre, heterozygous parental cell in light brown, OvoD mutant cells in light grey, fand mutant clone cells in pink.

    Flies were raised using standard techniques at 25 °C unless indicated. All crosses were set up in bottles and flipped to new food vials on a daily basis. The production of germline clones of the fandango allele was based on two crosses:
    1. The objective of the first cross is to generate the so-called “OvoD males” carrying the hs-flipase, the FRT 42B, the dominant female sterile OvoD mutation and a balancer CyO bearing the heat-shock-inducible pro-apoptotic transgene head involution defective (hid) (OvoD males genotype: y w hs-FLP22/Y; FRT 42B OvoD/CyO hs-hid). These males are produced by crossing virgins of the genotype y w hs-FLP22; If/CyO hs hid, with males of the genotype FRT 42B OvoD/T(1;2)OR64/CyO (see Figure 5 cross b1).
      Note: These males are easily recognized by the orange color of their eyes and by their curly wings (CyO).


      Figure 5. Genetic crosses to recover FRT/FLP induced female flies carrying germline mutant clones

    2. The objective of the second cross is to generate females carrying clonal germline mutants for the fandango allele.
    3. For this, virgins carrying the fandango mutant allele (genotype: w; FRT 42B, fand/CyO) were crossed with previously produced “OvoD males”.
    4. By the late second/third larval instar stages, the F1 progeny was heat-shocked in a 37 °C water bath for 1 h, in order to induce mitotic recombination and the lethality of organisms carrying the balancer (see Figure 5 cross b2).
    5. Finally, F1 adult females with normal wings (lack of the CyO balancer) were transferred to food vials supplemented with fresh baker’s yeast for 3 days prior to egg collection. Since OvoD is a dominant mutation that critically disrupts oogenesis, heterozygous OvoD females develop atrophic ovaries incapable of producing eggs. Accordingly, only the germline stem cells in which FRT-mediated mitotic recombination was induced by the flipase (hs-FLP) will be capable of producing mature eggs. These eggs will necessarily be homozygous for the fandango mutation and can thus be collected, processed and analyzed in order to characterize the embryogenesis phenotypes of the mutation (Figure 6C-D). As controls, germline clones without any associated mutations were generated by crossing virgin flies carrying only the FRT42B recombination site (genotype: w; FRT 42B/CyO) with “OvoD males”, followed by the aforementioned heat shock procedure (Figure 6A-B).
      Note: The number of flies used in the cross referred in section 5 are approximately 50 female virgins with 10 males.


      Figure 6. fandango maternal mutant embryos laid by females carrying germline clones originated by the FRT/FLP OvoD system. Panels show blastoderm cellularized control embryos (hs-FLP; FRT42B), and fand germline clone mutant embryos (hs-FLP; FRT42B fand, maternal mutant) (A-D). Control embryos showed normal epithelial architecture with elongated nuclei and columnar cell shape (A, B). fand germline clone mutant embryos showed abnormal epithelial architecture, the cortical nuclei failed to elongate and became mislocalized (C, D). (B-D) Magnification of C and D respectively. Embryos were stained for Slam (green), Neurotactin (red), and DNA (blue).

Recipes

  1. Fly food (1 L)
    Molasses (g): 80
    Beet syrup (g): 22
    Cornmeal (g): 80
    Yeast granulated (g): 18
    Soy flour (g): 10
    Agar (g): 8
    Boiling water (ml): 980
    15% Niapagin (ml): 12
    Propionic acid (ml): 8
    Weigh all ingredients except Niapagin and Proprionic acid in a plastic beaker
    Mix all ingredients in a beaker before adding to boiling water
    Add boiling water gradually
    Transfer the solution to a bottle
    Autoclaved at 121 °C, 30 min
    When the medium temperature reaches 45 °C-50 °C add Niapagin and Proprionic acid

Acknowledgments

We like to thank Paulo Navarro-Costa for critical reading of manuscript and Rui Martinho for his supervision. Funding: FCT-Fundacao para a Ciencia e Tecnologia (Portugal): Leonardo Gastón Guilgur, SFRH/BPD/47957/2008. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

References

  1. Chou, T. B. and Perrimon, N. (1992). Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila. Genetics 131(3): 643-653.
  2. Golic, K. G. and Lindquist, S. (1989). The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59(3): 499-509.
  3. Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181.
  4. Theodosiou, N. A. and Xu, T. (1998). Use of FLP/FRT system to study Drosophila development. Methods 14(4): 355-365.
  5. Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117(4): 1223-1237.

简介

FLP / FRT系统是基于将重组酶(翻转酶-FLP)靶向指定为翻转酶识别靶标(FRT)位点的特定DNA区域的定点重组技术。最初在酿酒酵母中鉴定,酵母FLP酶及其FRT重组靶点成功转移到果蝇中的每个主要染色体臂(Golic和Lindquist,1989)。这提供了以受控方式在发育过程中在体内介导有丝分裂重组的能力[在Theodosiou和Xu(1998)中修订]。有丝分裂重组事件的受控诱导通常通过在热休克(hs)启动子的控制下表达FLP来进行。这允许在特定发育时间窗口表达高FLP水平。携带这些遗传标记的FLP / FRT染色体的菌株大大增强了我们在种系和体细胞果蝇组织中研究基因功能的能力。在这里我们描述两种不同的方案:一种用于诱导和鉴定卵巢中的纯合突变体克隆,另一种用于产生雌性种系突变体,用于分析母体对胚胎发生的影响。

关键字:果蝇, FLP/FRT, 有丝分裂重组, 生殖系

材料和试剂

  1. 飞行股票
    1. w; FRT 42B,fand/CyO (Guilgur ,2014)
    2. y w hs-FLP 22 /Y; FRT 42B nls-GFP/CyO hs-hid(Guilgur et al。,2014)
    3. y w hs-FLP 22 ; If/CyO hs hid (Guilgur ,2014)
    4. /T(1; 2)OR64/CyO (Guilgur < ,2014; Bloomington Drosophila Stock Center,目录号:4434)
    5. 材料和试剂

      1. 飞行股票
        1. w; FRT 42B,fand/CyO (Guilgur ,2014)
        2. y w hs-FLP 22 /Y; FRT 42B nls-GFP/CyO hs-hid(Guilgur et al。,2014)
        3. y w hs-FLP 22 ; If/CyO hs hid (Guilgur ,2014)
        4. /T(1; 2)OR64/CyO (Guilgur < ,2014; Bloomington Drosophila Stock Center,目录号:4434)
        5. ...
        6. Napagin (Dutscher, catalog number: 789063)
        7. Propionic acid (Acros Organics)
        8. Fly food (see Recipes)

        Equipment

        1. Plastic bottles
        2. 37℃水浴
        3. 立体显像

        程序

        1. 使用FLP/FRT系统产生和标记突变体克隆
          在本部分中,我们描述了用于隐性突变fandango(fand)的纯合突变体克隆的生成,其已在Guilgur等人(2014)中描述。为了区分纯合突变体和野生型克隆,我们将FLP/FRT系统与细胞自主标记物组合,如最初在Xu和Rubin(1993)中描述的。我们使用标记有核定位信号(nls-GFP)的绿色荧光蛋白以便容易地基于存在(野生型克隆-nls-GFP加)来鉴定克隆或缺少(突变体克隆-nls-GFP减去)核GFP信号的(图1)。
          使用标准技术在25℃下饲养果蝇,除非另有说明。所有的十字架都设置在瓶子里,每天翻转到新的食物瓶至少4次
          1. 为了产生fandango 等位基因的纯合镶嵌克隆,我们杂交 携带突变等位基因(基因型: w; FRT 42B,fand/CyO )的virgins  携带以下标记的雄性:hs-翻译酶,FRT 42B,nls-GFP 构建体和携带热休克诱导型 促凋亡转基因<111>头转换缺陷( hid )(基因型: y w hs-FLP 22 /Y; FRT 42B nls-GFP /CyO hs-hid )。反向交叉也可以  (见图2)。
          2. 由晚二/三龄幼虫 阶段将F sub子代在37℃水浴中热休克1小时,在  以诱导有丝分裂重组和有机体的致死率 携带具有 转基因的平衡器。另外1小时热休克 在第二天可以做到增加克隆的频率 注意:选择发育阶段以诱导有丝分裂 通过热休克的重组可以根据待组织而变化 分析。连续两次热休克可能增加的速率 作为更多的翻译酶表达的结果,因此 将产生更多的克隆。然而,重要的是要注意 健康的原始股票在交叉使用(有时突变 股票疲软)和一个以上的热休克可能是有害的 苍蝇。在我们的fand等位基因的情况下,足够的热休克。  在第1节中提到的十字中使用的苍蝇的数量是 约15个女性处女与5个男性。


            图1.生成 并使用FLP/FRT系统和细胞标记物标记突变体克隆。在a 杂合亲本细胞(A),FLP诱导有丝分裂重组 在同源2R染色体臂上的FRT位点(黄色箭头)之间。 重组染色体在有丝分裂中的分离产生两个女儿 细胞:具有两个拷贝的突变等位基因的突变体细胞(D) 和仅含有野生型形式的基因(C)的野生型细胞。  细胞标记物nls-GFP 与野生型基因(野生型)共分离 类型双点克隆 - nls-GFP 加上) ( fand突变体克隆 nls-GFP 减去)。的 非重组细胞也由来自仅较轻的信号鉴定  一个 nls-GFP (B)(野生型一点克隆 - nls-GFP 。 在果蝇卵巢中的克隆马赛克的示意图(E)。 FLP重组酶靶序列(FRT)以橙色中的青色,fandango 突变和赭石中的nls-GFP 亲本细胞为浅棕色,单斑细胞为浅绿色,双斑 绿色的细胞,粉红色的 fand 突变体克隆细胞

            图2.遗传杂交以回收由细胞标记物标记的FRT/FLP诱导的克隆

          3. F 1成年雌性具有正常的翅膀(没有卷曲翅膀指示  缺少CyO 平衡器)转移到补充的食物小瓶中 与新鲜面包酵母在卵巢解剖前3天 处理(图3A-B)

            图3. Fandango 突变体克隆 卵巢。 卵巢发育在fandango突变体卵巢克隆(A,B)中是正常的。 缺少内源性nls-GFP ( nls-GFP 减去)表明细胞 对于fand 突变是纯合的。相反,存在内源性nls-GFP ( nls-GFP 加上)表示野生型克隆。卵巢染色 对于F-肌动蛋白(红色)和WGA(蓝色)。

        2. 使用FLP/FRT Ovo D 系统从种系突变体克隆产生fandango母体突变体胚胎
          在本节中,我们描述了雌性生殖系突变体克隆的生成以表征隐性合子致死突变fandango的母体效应(Guilgur等人,2014)。在该测定中应用的遗传技术利用了酵母"FLP/FRT"位点特异性重组系统与种系依赖性显性雌性不育Ovo D的组合的性质 突变[最初在Chou和Perrimon(1992)中描述](图4)。


          图4.使用FLP/FRT和Ovo 系统产生种系突变体克隆。 在杂合亲本细胞(A)中,FLP在同源2R染色体臂上的FRT位点(黄色箭头)之间诱导有丝分裂重组。重组染色体在有丝分裂时的分离产生两个子细胞:具有两个拷贝的突变等位基因的突变生殖细胞(D)和仅含有基因的野生型形式的野生型细胞C)。种系依赖性优势雌性不育Ovo D 突变的存在阻断产生萎缩性卵巢的卵子发生(B,C)。因此,所有发育的卵巢对于fand 等位基因是纯合突变体。在果蝇胚发生(E)期间种系克隆卵巢的生成的示意图。 FLP重组酶靶序列(FRT)以橙色中的青色,fandango 突变, Ovo D 在浅灰色突变体细胞中的浅褐色突变细胞中的杂合突变细胞中的突变细胞粉红色的细胞
          使用标准技术在25℃下饲养果蝇,除非另有说明。所有的十字架都设置在瓶子里,每天翻转到新的食物小瓶。 fandango 等位基因的种系克隆的产生基于两个杂交:
          1. 第一个杂交的目的是产生携带hs-翻译酶的所谓"Ovo D 雄性",FRT 42B,优势雌性不育Ovo 突变和携带热休克诱导的平衡器 促细胞凋亡转基因( )( ) 基因型: y w hs-FLP 22 FRT 42B Ovo D /CyO hs-hid )。这些男性是 通过交叉基因型 y w hs-FLP 22 的virgins产生; If/CyO hs 与基因型男性FRT 42B Ovo D /T(1; 2)OR64/CyO /em>(参见图5  cross b 1 )。
            注意:这些男性很容易被他们眼睛的橙色和他们的卷曲翅膀(CyO)识别。


            图5.恢复携带种系突变体克隆的FRT/FLP诱导的雌性果蝇的遗传杂交

          2. 第二个杂交的目的是产生携带fandango 等位基因的克隆种系突变体的雌性。
          3. 为此,携带fandango突变体等位基因的virgins(基因型: FRT 42B,fand/CyO )与先前产生的"Ovo D
          4. 通过晚第二/第三幼虫期,F 1子代 在37℃水浴中热休克1小时,以诱导有丝分裂 重组和携带平衡器的生物的致死性(参见 图5交叉b 2 )。
          5. 最后,具有正常翅膀的F 1 1成年雌性 (缺少CyO 3平衡器)转移到补充的食物小瓶中 与新鲜的面包酵母在收集鸡蛋前3天。由于Ovo D 是严重破坏卵子发生的显性突变,杂合子Ovo /em> 女性发展不能产生卵的萎缩性卵巢。 因此,只有生殖系干细胞其中FRT介导的有丝分裂 通过翻译酶(hES-FLP )能够诱导重组 产生成熟鸡蛋。这些卵必然对于fandango突变是纯合的,因此可以被收集,处理和分析 以表征突变的胚胎发生表型 (图6C-D)。作为对照,没有任何相关的种系克隆 突变通过交叉只携带的纯种苍蝇而产生 与"Ovo D 男性"的FRT42B重组位点(基因型: w; FRT 42B/CyO 随后进行上述热休克过程(图6A-B)。
            注意:第5节中提到的十字交叉中使用的苍蝇数量约为50个女性,有10个男性。


            图6. fandango 母体突变体胚胎由女性携带 源自FRT/FLP 系统的胚系克隆。 胚泡细胞化对照胚胎(hs -FLP; FRT42B)和胚胎克隆突变体胚胎(hs -FLP; FRT42B ,母体突变体) (广告)。对照胚胎显示正常的上皮结构 细长核和柱状细胞形状(A,B)。 fand 种系克隆 突变体胚胎显示异常上皮结构,皮层 核不能伸长并且变得错位(C,D)。 (B-D) 分别放大C和D.胚胎染色Slam (绿色),神经营养因子(红色)和DNA(蓝色)。

        食谱

        1. 飞食(1升)
          糖蜜(g):80
          甜菜糖(g):22
          玉米粉(g):80
          酵母颗粒(g):18
          大豆粉(g):10
          琼脂(g):8
          沸水(ml):980
          15%Niapagin(ml):12
          丙酸(ml):8
          在塑料烧杯中称量除Niapagin和Proprionic酸之外的所有成分 将所有成分在烧杯中混合后加入沸水中
          逐步加入沸水
          将溶液转移到瓶子
          121℃,30分钟,高压灭菌
          当介质温度达到45℃-50℃时,加入Niapagin和丙氨酸

        致谢

        我们要感谢保罗·纳瓦罗 - 科斯塔对手稿的批判性阅读,并感谢他的监督。 资金:FCT-Fundacao para a Ciencia e Tecnologia(葡萄牙):LeonardoGastónGuilgur,SFRH/BPD/47957/2008。 资助者在研究设计,数据收集和解释或提交出版工作的决定方面没有任何作用。

        参考文献

        1. Chou,T.B.and Perrimon,N。(1992)。 使用酵母位点特异性重组酶在果蝇中产生雌性种系嵌合体。 Genetics 131(3):643-653。
        2. Golic,K.G.and Lindquist,S。(1989)。 酵母的FLP重组酶催化果蝇基因组中的位点特异性重组。 Cell 59(3):499-509。
        3. Guilgur,L.G.,Prudencio,P.,Sobral,D.,Liszekova,D.,Rosa,A.and Martinho,R.G。(2014)。 在果蝇早期胚胎发育过程中需要高效的前mRNA剪接。 3:e02181。
        4. Theodosiou,N.A。和Xu,T。(1998)。 使用FLP/FRT系统研究果蝇的发育。 方法 14(4):355-365。
        5. Xu,T。和Rubin,G.M。(1993)。 分析发育中和成年果蝇组织中的遗传马赛克。 Development 117(4):1223-1237。
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Copyright Prudêncio and Guilgur. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Prudêncio, P. and Guilgur, L. G. (2015). FLP/FRT Induction of Mitotic Recombination in Drosophila Germline. Bio-protocol 5(9): e1458. DOI: 10.21769/BioProtoc.1458.
  2. Guilgur, L. G., Prudencio, P., Sobral, D., Liszekova, D., Rosa, A. and Martinho, R. G. (2014). Requirement for highly efficient pre-mRNA splicing during Drosophila early embryonic development. Elife 3: e02181.
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