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Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes
疟蚊多线染色体的荧光原位杂交   

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Abstract

Fluorescence in situ hybridization (FISH) is a method that uses a fluorescently labeled DNA probe for mapping the position of a genetic element on chromosomes. A DNA probe is prepared by incorporating Cy-3 or Cy-5 labeled nucleotides into DNA by nick-translation or a random primed labeling method. This protocol was used to map genes (Sharakhova et al., 2010) and microsatellite markers (Kamali et al., 2011; Peery et al., 2011) on polytene chromosomes from ovarian nurse cells and salivary glands of malaria mosquitoes. Detailed physical genome mapping performed on polytene chromosomes has the potential to link DNA sequences to specific chromosomal structures such as heterochromatin (Sharakhova et al., 2010). This method also allows comparative cytogenetic studies (Sharakhova et al., 2011; Xia et al., 2010), and reconstruction of species phylogenies (Kamali et al., 2012).

Keywords: Mosquito(蚊子), Chromosome(染色体), Mapping(映射), Genome(基因组), FISH(鱼)

Materials and Reagents

  1. Early fourth instar Anopheles larvae
  2. Female Anopheles mosquitoes
  3. Template DNA
  4. Fisherfinest* Premium Extra-Thick Frosted Microscope Slides (Double frosted coating) (Thermo Fisher Scientific, catalog number: 12-544-6 )
  5. Fisherfinest* Premium Cover Glasses (22 x 22 mm) (Thermo Fisher Scientific, catalog number: 12-544-10 )
  6. 50% Propionic acid in water
  7. Razor blade
  8. Liquid nitrogen
  9. Ethanol, molecular biology grade
  10. Microscope slide staining jar with lid
  11. Random Primed DNA Labeling Kit (Roche Applied Science, catalog number: 11004760001 )
  12. Random Primers DNA Labeling System (Life Technologies, InvitrogenTM, catalog number: 18187-013 )
  13. Formamide (Super pure) (Fisher Bioreagent, catalog number: BP228-100 )
  14. Dextran Sulfate Sodium salt from Leuconostoc spp. (Sigma-Aldrich, catalog number: D8906 )
  15. Prolong® Gold antifade reagent (Life Technologies, InvitrogenTM, catalog number: P36930 )
  16. Cy3-dUTP (GE Healthcare, catalog number: PA53022 )
  17. Cy5-dUTP (GE Healthcare, catalog number: PA55022 )
  18. YOYO®-1 Iodide (491/509)–1 mM Solution in DMSO (Life Technologies, InvitrogenTM, catalog number: Y3601 )
  19. Paraformaldehyde (Sigma-Aldrich, catalog number: F8775 )
  20. DNA polymerase I (Fermentas, catalog number: EP0041 )
  21. DNase I (Fermentas, catalog number: EN0521 )
  22. QIAquick® Gel Extraction Kit (QIAGEN, catalog number: 28704 )
  23. QIAquick® PCR purification Kit (QIAGEN, catalog number: 28104 )
  24. Carnoy's solution (see Recipes)
  25. 20x SSC (see Recipes)
  26. 3 M NaAC (see Recipes)
  27. 1x PBS (see Recipes)
  28. Hybridization buffer (see Recipes)

Equipment

  1. 1.5 ml microcentrifuge tubes
  2. Forceps
  3. Disposable transfer pipette
  4. Dissecting needles
  5. Research stereo microscope (Leica, model: VA-OM-E194-354 )
  6. Phase contrast compound microscope with 10x, 20x, 40x and 100x objective lenses
  7. Thermal cycler
  8. Vacufuge® vacuum concentrator (Eppendorf, model: 022820001 )
  9. Incubator
  10. Water Bath
  11. Vortexer
  12. Confocal Microscope or Fluorescence Microscope

Procedure

  1. Polytene chromosome preparation
    A-1   Salivary gland chromosome preparation
    1. Preserve early fourth instar larvae in Carnoy's Solution and keep at -20 °C.
    2. Remove one fourth-instar larva from the vial with a pair of forceps and place it on a dust-free microscope slide with back upward, then put a drop of fresh Carnoy's solution onto it immediately (Figure 1a).
      Note: Continue adding drops of Carnoy's solution when needed to prevent drying out until dipping 50% Propionic acid onto the gland.
      While firmly holding the larva with one dissecting needle, gently pull the head away from thorax with another needle (Figure 1b). Insert a needle from the middle rear of the thorax just underneath the cuticle, and gently move forward to break the thorax cuticle along the mid dorsal line (Figure 1c). Carefully open up the thorax and separate the salivary gland from connecting tissue (Figure 1d and 1e). Remove the carcass and other tissue from slide and put one drop of fresh 50% Propionic acid onto the gland (Figure 1f).


      Figure 1. Dissection of salivary glands in 4th instar larva of An. Sinensis

    3. Cover gland with a dust-free coverslip and leave them for about 5 min. After 5 min, place a piece of filter paper over the coverslip, hold the four edges still with fingers. Gently tap it with a pencil eraser to release the polytene chromosomes from the salivary gland.
    4. Examine the banding pattern and spread of polytene chromosomes using a phase-contrast microscope.
    5. Place slides with good chromosomal preparations in a humid chamber with 4x SSC in the bottom of the chamber, at 60 °C for 15-20 min. After heating, put slides at 4 °C overnight or until immersing the slides in liquid nitrogen. Heating can be done on the Thermobrite machine with the absorbent strips soaked in distilled water.
      Note: Slides can dry out if left at 4 °C for extended periods of time. Leaving them at 4 °C for longer than overnight is not recommended.
    6. While holding one corner of the slide with forceps or a gloved hand, dip chromosome preparation into the liquid nitrogen so that the coverslip is completely immersed. Hold slide in liquid nitrogen until the bubbling stops (usually 10-15 sec). Take it out of the liquid nitrogen and immediately remove the coverslip with a razor blade from one corner. It sometimes helps to put the slide on a flat surface when trying to remove the cover slip. Put slide in a slide jar with prechilled 50% Ethanol (-20 °C) and keep at 4 °C for at least 2 h.
    7. Dehydrate the preparations in slide jar with an ethanol series of 70%, and 90% for 5 min each at 4 °C and then 100% ethanol for 5 min at room temperature. Air dry, and keep slides in slide box until ready for use in in situ hybridization (Figure 2).
      Note: Slides that are kept protected from dust and debris can be used for FISH at least within a year after the preparations are made.


      Figure 2. Polytene chromosomes from salivary glands of An. sinensis

    A-2  Chromosome preparation from Anopheles ovaries
    1. Dissect the ovaries of Anopheles mosquitoes from half gravid females 18-33 h after 2nd or 3rd blood feeding (Christophers' III stage) and keep 4-5 ovaries in a vial with 1 ml of Carnoy's sollution. After fixing the ovaries for 24 h at room temperate, transfer the vials to -20 °C for storage.
      Note: Females should be bloodfed and lay eggs at least once before bloodfeeding again and dissecting ovaries for chromosomal preparations.
    2. To make the chromosome preparations, take one ovary out of the vials with a pair of forceps (or a transfer pipet) and place it into a drop of Carnoy's solution on microscope slide. After carefully removing tissues, trachea and blood, quickly separate the follicles from one ovary into 2-4 pieces. Up to four preparations can be made from one ovary.
      Note: While dissecting, the ovaries should never be allowed to dry. Continue adding drops of Carnoy's solution when needed to prevent drying of the ovaries.
    3. On 4-8* microscope slides, add each of the pieces of divided ovary and one drop of 50% propionic acid on a separate slide. Let the pieces of ovary rest in propionic acid for about 5 min until follicles become clear, and swell to about twice their original size.
      *number of slides you need depends of how many pieces each ovary is divided into.
    4. For each slide, use a dissecting microscope to separate the cleared follicles from each other and any other tissue or debris on the slide. Remove tissue and debris by wiping it away with a piece of paper towel, and apply a fresh drop of 50% propionic to the separated follicles.
    5. Do the same as steps 3-7 in "Salivary gland chromosome preparation".

  2. Probe preparation and labeling
    If using PCR products as probe, purify the PCR product from an agarose gel or from the PCR reaction using a QIAquick® Gel Extraction Kit or QIAquick® PCR purification Kit. Similar kits that remove excess nucleotides can also be used. However, when using a kit, dissolve the DNA in double distilled water instead of the elution buffer suggested in the final step.
    B-1 Random Primer labeling protocol for fragments shorter than 1 kb (Random Primed DNA Labeling kit from Roche
    1. Add 25 ng template DNA into double distilled water to a final volume of 13.5 μl in a microcentrifuge tube.
    2. Denature the DNA by heating in a boiling water bath for 10 min at 95 °C and chilling quickly in an ice bath.
    3. Add the following to the freshly denatured probes on ice:
      dGTP, 1.0 mM
      1 μl
      dCTP, 1.0 mM
      1 μl
      dATP, 1.0 mM
      1 μl
      Reaction Mixture (Vial 6)
      2 μl
      Klenow enzyme (Vial 7)
      1 μl
      Cy3 or Cy5-dUTP, 1.0 mM
      0.5 μl
      Mix and centrifuge briefly.
    4. Incubate for 1 h to 20 h (overnight) at 37 °C.
    5. Add 1/10 volume of 3 M NaAC and 2.5-3 volume of 100% ethanol. And mix by inverting the tubes. Keep at -80 °C or -20 °C for at least 3 h or until probes are needed for hybridization. If necessary, probes can be left in the freezer for long-term storage.
    B-2 Random Primer labeling protocol for fragments shorter than 1 kb (Random Primers DNA Labeling System from Invitrogen)
    1. Mix 1 μl DNA and 10 μl 2.5x Random Primer Solution and 2.5 μl sterile water well.
    2. Denature 5 min in boiling water or heating block, immediately cool on ice.
    3. Add 1.25 μl 1.0 mM dNTP mix (without a labeled dNTP), 8.75 μl water and 1 μl Klenow Fragment, mix gently but thoroughly.
    4. Add 0.5 μl Cy3 or Cy5-dUTP fluorescent nucleotide to each tube, when finished, tube must be covered immediately to protect from light. Mix well and incubate at 37 °C for 1.5 h.
    5. Do the same as step 5 in Section II-1 "Random Primer labeling protocol for fragments shorter than 1 kb (Random Primed DNA Labeling kit from Roche)".
    B-3 Nick Translation labeling for fragment longer than 1 kb (1-150 kb)
    1. Prepare the following reaction mixture on ice:
      10x buffer for DNA Polymerase I
      5 μl
      1.0 mM dATP, dCTP, dGTP and 0.3 mM dTTP mixture
      5 μl
      DNase I freshly diluted to 0.02 units/μl
      4 μl**
      DNA Polymerase I
      1 μl**
      Template DNA
      1 μg
      Cy3- or Cy5-dUTP
      1 μl
      BSA diluted to 0.5 mg/ml
      5 μl
      Add water to final volume 50 μl
      Note: This protocol can be scaled down by 1/2 to accommodate 500 ng of template DNA.
      **Final concentrations of DNase I and DNA Polymerase I have to be optimized based on factors including initial size of template DNA, template DNA concentration and reaction time. Larger template size and greater template concentration generally require more DNase.
    2. Incubate the mix at 15 °C for 2-3 h.
    3. Run 3 μl of reaction mixture on an agarose gel to determine the size of digested fragments. Fragments should be 100-600 bp for best hybridization results. If fragments are still larger than this, incubate at 15 °C for additional time.
    4. To terminate the reaction and precipitate labeled probes, do the same as step 5 in Section B-2 "Random Primer labeling protocol for fragments shorter than 1 kb (Random Primed DNA Labeling kit from Roche)"
      Note: Fluorescently labeled probes should be protected from light! In the steps following, even where it is not explicitly stated, make efforts to protect probes from light.
  3. Chromosomal fixation
    1. Do step C-2 and C-3 if slides are more than two months old. Otherwise, go to step C-4.
    2. Fix slides in 1:3 glacial acetic acid: methanol at RT for 10 min and air-dry.
    3. Dehydrate slides in 100% ethanol for 10 min and air dry again.
    4. Immerse slides in 1x PBS for 20 min at RT.
    5. Fix slides at room temperature in 4% paraformaldehyde for 1 min.
      Note: Paraformaldehyde is hazardous and should be handled carefully. Avoid breathing gas or dust during preparation: wear gloves and other PPE when handling. Paraformaldehyde solution should not be dumped down drains.
    6. Dehydrate the slides through an ethanol series of 50%, 70%, 90%, 2x 100% for 5 min each at RT.
    7. Air-dry the slides.

  4. In situ hybridization
    1. Centrifuge the tubes of labeled probes at 20,817 x g for 10 min. Carefully remove the supernatant and vacumfuge the tubes for 20 min to dry pellets.
    2. Dissolve dry probes in hybridization buffer prewarmed to 37 °C. The amount of hybridization buffer used to dissolve depends on the total amount of DNA you are dissolving. Dissolve 1 μg of DNA in 20-40 μl of warmed hybridization buffer.
    3. In a clean microcentrifuge tube, combine at least 250 ng each of one blue (Cy5 labeled) and one red (Cy3 labeled) probes. In situ hybridization is efficient if at least 500 ng of DNA is hybridized on the slide. Vortex and centrifuge the tube of combined probe briefly.
    4. Transfer the above prepared solution of combined probes to a chromosome preparation slide and cover with a 22 x 22 mm coverslip. Remove any large air bubbles with gentle pressure.
    5. Denature the target and probe DNA by placing the slides on the Thermobrite machine at 90 °C for 10 min. Thermobrite machine does not need to be humid.
    6. Seal edges of cover slip with rubber cement.
    7. Transfer the slides to pre-warmed humid chambers with 4x SSC at the bottom of the chambers and incubate at 39 °C for interspecies (e.g An. gambiae probe to An. stephensi chromosomes) or 42 °C for intraspecies hybridization for 3-18 h (usually overnight).
      Note: Because there are fluorescently labeled probes on the slide, humid chambers should be impermeable to light.

  5. Washing
    1. Carefully remove rubber cement with forceps and coverslip.
    2. In a slide jar covered with aluminum foil, wash the slides with 1x SSC at 39 °C after interspecies or 0.2x SSC at 42 °C after intraspecies hybridization for 20 min in 50 ml without shaking.
    3. Wash the slides with 1x SSC after interspecies or 0.2x SSC after intraspecies hybridization at RT for 20 min in 50 ml without shaking.
    4. Dilute fluorescent dye YOYO-1 100 times in 1x PBS to make a stock solution. Mix 10 μl of 100x diluted YOYO-1 with 90 μl 1x PBS for each slide that you want to stain. The working solution of YOYO-1 is 1,000x diluted relative to original concentration.
    5. After washing in SSC for 20 min at room temperature, rinse slide in 1x PBS, and add 100 μl of YOYO-1 in PBS on each slide. Cover with parafilm. Leave at RT for 10 min inside of a slide box or somewhere dark.
    6. Rinse in 1x PBS and add 10 μl Prolong Gold antifade reagent, place coverslip on slide and blot out bubble. Keep in the slide box at 4 °C.

  6. Signal detection
    Detect the signals using a confocal or fluorescence microscope and map them to the polytene chromosomes of Anopheles mosquitoes (Figure 3).


    Figure 3. Fluorescence in situ hybridization and mapping of DNA probes on polytene chromosomes from ovarian nurse cells of An. stephensi

Recipes

  1. Carnoy's solution: Methanol:Glacial Acetic Acid = 3:1
    Note: Carnoy's solution should be used with good ventilation or in a fume hood. Gloves should also be worn while using Carnoy's solution and it should not be disposed of down the drain.
  2. 1x PBS (1 L)
    NaCl
    8.01 g
    KCl
    0.20 g
    NaH2PO4 (anhydrous)
    1.15 g
    KHP2O4 (anhydrous)
    0.20 g
  3. 20x SSC (500 ml)
    Sodium chloride
    87.5 g
    Sodium citrate
    44 g
    Add 1 N HCl to pH 7.0
  4. Hybridization buffer (2 ml)
    20x SSC
    120 μl
    Dextran sulfate
    0.2 g
    Formamide
    1.2 ml
    Water
    580 μl
  5. 3 M NaAC
    Dissolve 24.61 g of Sodium Acetate (anhydrous) in 100 ml water.

Acknowledgments

The protocol was adapted from previously published papers: Sharakhova et al. (2010) and Kamali et al. (2012). We thank the Chinese Centre for Disease Control and Prevention, Shanghai, China for providing an Anopheles sinensis colony. This work was supported by a National Natural Science Foundation of China (31301877) to AX and by a National Institutes of Health (Bethesda, MD, U.S.A.) grant (5R21AI094289) to IVS. AP and IVS were supported in part by the Institute for Critical Technology and Applied Science (ICTAS) and the NSF award 0850198.

References

  1. Kamali, M., Sharakhova, M. V., Baricheva, E., Karagodin, D., Tu, Z. and Sharakhov, I. V. (2011). An integrated chromosome map of microsatellite markers and inversion breakpoints for an Asian malaria mosquito, Anopheles stephensi. J Hered 102(6): 719-726.
  2. Kamali, M., Xia, A., Tu, Z. and Sharakhov, I. V. (2012). A new chromosomal phylogeny supports the repeated origin of vectorial capacity in malaria mosquitoes of the Anopheles gambiae complex. PLoS Pathog 8(10): e1002960. 
  3. Peery, A., M. V. Sharakhova, Antonio-Nkondjio, C., Ndo, C., Weill, M., Simard, F., and I. V. Sharakhov. 2011. Improving the population genetics toolbox for the study of the African malaria vector Anopheles nili: microsatellite mapping to chromosomes. Parasites and Vectors 4:202. 
  4. Sharakhova, M. V., Xia, A., Tu, Z., Shouche, Y. S., Unger, M. F. and Sharakhov, I. V. (2010). A physical map for an Asian malaria mosquito, Anopheles stephensi. Am J Trop Med Hyg 83(5): 1023-1027. 
  5. Sharakhova, M. V., George, P., Brusentsova, I. V., Leman, S. C., Bailey, J. A., Smith, C. D. and Sharakhov, I. V. (2010). Genome mapping and characterization of the Anopheles gambiae heterochromatin. BMC Genomics 11: 459.
  6. Sharakhova, M. V., Antonio-Nkondjio, C., Xia, A., Ndo, C., Awono-Ambene, P., Simard, F. and Sharakhov, I. V. (2011). Cytogenetic map for Anopheles nili: application for population genetics and comparative physical mapping. Infect Genet Evol 11(4): 746-754..
  7. Xia, A., Sharakhova, M. V., Leman, S. C., Tu, Z., Bailey, J. A., Smith, C. D. and Sharakhov, I. V. (2010). Genome landscape and evolutionary plasticity of chromosomes in malaria mosquitoes. PLoS One 5(5): e10592 

简介

荧光原位杂交(FISH)是一种使用荧光标记的DNA探针来绘制遗传元件在染色体上的位置的方法。 通过缺口翻译或随机引物标记法将Cy-3或Cy-5标记的核苷酸掺入DNA中来制备DNA探针。 该方案用于将基因(Sharakhova等人,2010)和微卫星标记物(Kamali等人,2011; Peery等人, >,2011)对来自卵巢保护细胞和疟疾蚊子的唾液腺的多角体染色体。 在多聚染色体上进行的详细的物理基因组作图具有将DNA序列连接到特异性染色体结构(例如异染色质)的潜力(Sharakhova等人,2010)。 该方法还允许比较细胞遗传学研究(Sharakhova等人,2011; Xia等人,2010)和物种系统发育的重建(Kamali等人, ,2012)。

关键字:蚊子, 染色体, 映射, 基因组, 鱼

材料和试剂

  1. 早期四龄幼虫幼虫
  2. 女性蚊子
  3. 模板DNA
  4. Fisherfinest * Premium超厚磨砂显微镜幻灯片(双面磨砂涂层)(Thermo Fisher Scientific,目录号:12-544-6)
  5. Fisherfinest * Premium Cover Glasses(22 x 22 mm)(Thermo Fisher Scientific,目录号:12-544-10)
  6. 50%丙酸的水溶液
  7. 剃刀刀片
  8. 液氮
  9. 乙醇,分子生物学等级
  10. 带盖的显微镜载玻片染色瓶
  11. 随机引物DNA标记试剂盒(Roche Applied Science,目录号:11004760001)
  12. 随机引物DNA标记系统(Life Technologies,Invitrogen TM ,目录号:18187-013)
  13. 甲酰胺(超纯)(Fisher Bioreagent,目录号:BP228-100)
  14. 来自明串珠菌的硫酸葡聚糖钠盐。 (Sigma-Aldrich,目录号:D8906)
  15. Prolong Gold抗消色剂(Life Technologies,Invitrogen TM ,目录号:P36930)
  16. Cy3-dUTP(GE Healthcare,目录号:PA53022)
  17. Cy5-dUTP(GE Healthcare,目录号:PA55022)
  18. YOYO碘化物(491/509)-1mM DMSO溶液(Life Technologies,Invitrogen TM ,目录号:Y3601)
  19. 多聚甲醛(Sigma-Aldrich,目录号:F8775)
  20. DNA聚合酶I(Fermentas,目录号:EP0041)
  21. DNase I(Fermentas,目录号:EN0521)
  22. QIAquick 凝胶提取试剂盒(QIAGEN,目录号:28704)
  23. QIAquick PCR纯化试剂盒(QIAGEN,目录号:28104)
  24. Carnoy的解决方案(见配方)
  25. 20x SSC(请参阅配方)
  26. 3 M NaAC(见配方)
  27. 1x PBS(请参阅配方)
  28. 杂交缓冲液(参见配方)

设备

  1. 1.5 ml微量离心管
  2. 镊子
  3. 一次性转移移液器
  4. 解剖针
  5. 研究立体显微镜(Leica,型号:VA-OM-E194-354)
  6. 具有10x,20x,40x和100x物镜的相差化合物显微镜
  7. 热循环仪
  8. Vacufuge 真空浓缩器(Eppendorf,型号:022820001)
  9. 孵化器
  10. 水浴
  11. Vortexer
  12. 共焦显微镜或荧光显微镜

程序

  1. Polytene染色体制备
    A-1  唾液腺染色体制备
    1. 保存早期四龄幼虫在Carnoy的解决方案,并保持在-20°C。
    2. 用一把镊子从小瓶中取出一个四龄幼虫,并将其置于无背衬的显微镜载玻片上,向上,然后立即将一滴新鲜的卡诺溶液放在其上(图1a)。
      注意:当需要时,继续加入Carnoy's溶液滴以防止干燥,直到将50%丙酸浸渍到腺体上。
      在用一个解剖针牢固地握住幼虫的同时,用另一个针轻轻地将头部远离胸部(图1b)。从正好在角质层下面的胸部的中间后部插入针,并且轻轻地向前移动以沿着背中线断开胸部角质层(图1c)。小心地打开胸部,并从连接组织分离唾液腺(图1d和1e)。从幻灯片中取出尸体和其他组织,并将一滴新鲜的50%丙酸放在腺体上(图1f)。


      图1.在第4代晚期幼虫中的唾液腺的解剖。西安

    3. 用无尘盖玻片盖腺,并离开他们约5分钟。 5分钟后,将一张滤纸放在盖玻片上,用手指保持四个边缘静止。轻轻点击用铅笔橡皮擦释放多烯染色体从唾液腺。
    4. 使用相差显微镜检查聚苯染色体的条带模式和扩散。
    5. 将具有良好染色体制剂的载玻片置于在室底部具有4×SSC的潮湿室中,在60℃下15-20分钟。加热后,将载玻片在4℃下过夜或直到将载玻片浸没在液氮中。加热可以在Thermobrite机器上进行,吸收条带浸泡在蒸馏水中。
      注意:如果在4°C长时间放置,载玻片可能会变干。不建议将它们在4°C下放置超过隔夜。
    6. 用镊子或戴手套的手握住载玻片的一个角,将染色体制备物浸入液氮中,使得盖玻片完全浸没。在液氮中保持载玻片,直到鼓泡停止(通常10-15秒)。拿出液氮,立即用刀片从一个角落去除盖玻片。当试图取下盖玻片时,有时有助于将载玻片放在平坦的表面上。将载玻片置于带有预冷的50%乙醇(-20℃)的玻璃罐中并在4℃保持至少2小时。
    7. 在玻璃罐中的制剂用70%的乙醇系列和90%在4℃下各自5分钟,然后在室温下100%乙醇5分钟。空气干燥,并将载玻片保持在载玻片盒中,直到准备好用于原位杂交(图2)。
      注意:保护免受灰尘和碎屑影响的幻灯片可以在制作后至少一年内用于FISH。


      图2.来自from的唾液腺的多烯染色体。 sinensis

    A-2 来自卵巢的 染色体制备
    1. 在2次或3次血液喂养(Christophers'III期)后18-33小时从半妊娠女性解剖蚊子的卵巢保持4-5卵巢在小瓶与1毫升Carnoy的溶液。在室温下固定卵巢24小时后,将小瓶转移至-20°C储存 注意:女性应该进行血液喂养,并在再次进行供血之前至少产下一次卵,并切除卵巢进行染色体制备。
    2. 为了制备染色体,用一对镊子(或移液管)从小瓶中取出一个卵巢,并将其放入显微镜载玻片上的一滴Carnoy's溶液中。仔细去除组织,气管和血液后,迅速将卵泡从一个卵巢分离成2-4片。可以从一个卵巢制备多达四个制剂。
      注意:在解剖时,卵巢不应该被允许干燥。当需要时,继续加入Carnoy's溶液滴以防止卵巢干燥。
    3. 在4-8 *显微镜载玻片上,在单独的载玻片上加入每片分裂的卵巢和一滴50%丙酸。让卵巢的部分休息在丙酸约5分钟,直到毛囊变清澈,膨胀到原来的大小的两倍。
      *您需要的幻灯片数量取决于每个卵巢分成多少块。
    4. 对于每个幻灯片,使用解剖显微镜分离清除的卵泡彼此和幻灯片上的任何其他组织或碎片。通过用一块纸巾擦拭去除组织和碎片,并将新鲜的50%丙酸滴加到分离的卵泡中。
    5. 与"唾液腺染色体准备"中的步骤3-7相同。

  2. 探头准备和标签
    如果使用PCR产物作为探针,使用QIAquick凝胶提取试剂盒或QIAquick PCR纯化试剂盒从琼脂糖凝胶或PCR反应中纯化PCR产物。也可以使用除去过量核苷酸的类似试剂盒。然而,当使用试剂盒时,将DNA溶解在双蒸水中,而不是最终步骤中建议的洗脱缓冲液。
    B-1 用于短于1kb的片段的随机引物标记方案(来自Roche的随机引物DNA标记试剂盒
    1. 将25 ng模板DNA加入双蒸水中,至微量离心管中的最终体积为13.5μl
    2. 通过在95℃下在沸水浴中加热10分钟使DNA变性,并在冰浴中快速冷却。
    3. 在冰上加入新鲜变性的探针:
      dGTP,1.0mM
      1微升
      dCTP,1.0mM
      1微升
      dATP,1.0mM
      1微升
      反应混合物(小瓶6)
      2微升
      Klenow酶(Vial 7)
      1微升
      Cy3或Cy5-dUTP,1.0mM
      0.5μl
      混合并短暂离心。
    4. 在37℃下孵育1小时至20小时(过夜)
    5. 加入1/10体积的3M NaAC和2.5-3体积的100%乙醇。 并通过翻转管混合。 保持在-80°C或-20°C至少3小时或直到需要探针杂交。 如果需要,探针可以留在冷冻箱中长期储存
    B-2 用于短于1kb的片段的随机引物标记方案(来自Invitrogen的Random Primers DNA Labeling System)
    1. 混合1微升DNA和10微升2.5x随机引物溶液和2.5微升无菌水井
    2. 在沸水或加热块中变性5分钟,立即在冰上冷却
    3. 加入1.25μl1.0mM dNTP混合物(无标记的dNTP),8.75μl水和1μlKlenow片段,轻轻混合。
    4. 每管加入0.5μlCy3或Cy5-dUTP荧光核苷酸,完成后,必须立即覆盖管以防止光照。 充分混合并在37℃孵育1.5小时
    5. 与第II-1节"用于短于1kb的片段的随机引物标记方案(来自Roche的随机引物DNA标记试剂盒)"中的步骤5相同。
    B-3对于长于1kb(1-150kb)的片段的翻译标记
    1. 在冰上制备以下反应混合物:
      DNA聚合酶I的10x缓冲液
      5微升
      1.0mM dATP,dCTP,dGTP和0.3mM dTTP混合物 5微升
      新鲜稀释至0.02单位/μl的DNA酶 4μl**
      DNA聚合酶I
      1μl**
      模板DNA
      1微克
      Cy3-或Cy5-dUTP
      1微升
      BSA稀释至0.5mg/ml 5微升
      加水至最终体积50μl
      注意:此协议可以按 1/2 缩小以容纳500 ng的模板DNA。
      DNA酶I和DNA聚合酶I的最终浓度必须基于包括模板DNA的初始大小,模板DNA浓度和反应时间的因素进行优化。 较大的模板大小和较大的模板浓度通常需要更多的DNase。
    2. 在15°C孵育混合物2-3小时。
    3. 在琼脂糖凝胶上运行3μl反应混合物以确定消化的片段的大小。 片段应为100-600bp以获得最佳杂交结果。 如果碎片仍然大于此值,在15℃下再孵育一段时间
    4. 为了终止反应并沉淀标记的探针,进行与B-2节"用于短于1kb的片段的随机引物标记方案"(来自Roche的随机引物DNA标记试剂盒)中的步骤5相同的操作。
      注意:荧光标记的探针应该避光! 在接下来的步骤中,即使没有明确说明,也要努力保护探头免受光照。
  3. 染色体固定
    1. 如果载玻片超过两个月大,请执行步骤C-2和C-3。 否则,请转到步骤C-4。
    2. 将载玻片在1:3冰醋酸:甲醇中室温固定10分钟并风干
    3. 脱水在100%乙醇中滑动10分钟,再次风干
    4. 在室温下将载玻片浸入1x PBS中20分钟。
    5. 将载玻片在室温下在4%多聚甲醛中固定1分钟。
      注意:多聚甲醛是危险的,应小心处理。 避免在准备过程中呼吸气体或灰尘:处理时戴手套和其他PPE。 多聚甲醛溶液不应倾倒在下水道。
    6. 将载玻片在室温下通过50%,70%,90%,2x 100%的乙醇系列脱水,每次5分钟。
    7. 空气干燥载玻片。

  4. 原位杂交
    1. 在20,817×g离心标记的探针的管10分钟。小心地取出上清液,并抽空管子20分钟以干燥沉淀
    2. 将干探针溶解于预热至37℃的杂交缓冲液中。用于溶解的杂交缓冲液的量取决于要溶解的DNA的总量。将1μgDNA溶于20-40μl温热的杂交缓冲液中
    3. 在干净的微量离心管中,结合至少250ng的每个蓝色(Cy5标记的)和一个红色(Cy3标记的)探针。如果至少500ng的DNA杂交,原位杂交是有效的幻灯片。涡旋并短暂离心组合探头的管
    4. 将上述制备的组合探针溶液转移到染色体制备载玻片上,并用22×22mm盖玻片盖上。用温和的压力除去任何大气泡。
    5. 通过将载玻片置于Thermobrite机器上在90℃下10分钟来使靶标和探针DNA变性。 Thermobrite机器不需要潮湿。
    6. 用橡胶胶水密封盖玻片的边缘。
    7. 将载玻片转移到预热的湿室,在室底部用4×SSC,并在39℃下孵育用于种间(例如 冈比亚 Stephensi染色体)或42℃用于内部杂交3-18小时(通常过夜)。
      注意:因为幻灯片上有荧光标记的探针,潮湿的室应该是不透光的。

  5. 洗涤
    1. 使用镊子和盖玻片小心清除橡胶水泥
    2. 在用铝箔覆盖的玻璃罐中,在种子之后在39℃下用1x SSC洗涤载玻片,或在50℃下在不摇动的情况下在42℃下在0.2×SSC中杂交20分钟后洗涤载玻片。
    3. 在种子之后用1x SSC洗涤载玻片或在室温下在50ml不摇动的情况下在室内杂交20分钟后用0.2x SSC洗涤载玻片。
    4. 稀释荧光染料YOYO-1 100倍在1×PBS中制成储备溶液。混合10μl的100x稀释的YOYO-1与90μl1×PBS的每个幻灯片,你想染色。 YOYO-1的工作溶液相对于原始浓度稀释1,000倍。
    5. 在室温下在SSC中洗涤20分钟后,在1x PBS中漂洗载玻片,并在每个载玻片上加入100μl的YOYO-1 PBS。用石蜡膜覆盖。在室温下放置10分钟,在滑动盒内或某处暗处
    6. 冲洗1 x PBS,加入10μlProlong Gold抗衰减试剂,将盖玻片放在载玻片上,并吹出气泡。保持在4°C的滑动盒。

  6. 信号检测
    使用共聚焦或荧光显微镜检测信号,并将它们映射到按蚊蚊子的多染色体(图3)。


    图3.来自An的卵巢护士细胞的聚丙烯染色体上的DNA探针的荧光原位杂交和作图。 stephensi

食谱

  1. Carnoy溶液:甲醇:冰乙酸= 3:1
    注意:Carnoy的溶液应该在良好的通风或通风橱中使用。 使用Carnoy的溶液时,也应戴上手套,不应该在排水管下方处理。
  2. 1×PBS(1L)
    NaCl
    8.01克
    KCl
    0.20克
    NaH 2 PO 4(无水)
    1.15克
    (无水的)。
    0.20克
  3. 20x SSC(500ml)
    氯化钠
    87.5克
    柠檬酸钠
    44克
    加入1N HCl至pH7.0
  4. 杂交缓冲液(2ml)
    20x SSC
    120微升
    硫酸葡聚糖
    0.2 g
    甲酰胺
    1.2 ml

    580μl
  5. 3 M NaAC
    将24.61g乙酸钠(无水)溶于100ml水中

致谢

该方案改编自以前发表的文章:Sharakhova等人(2010)和Kamali等人(2012)。 我们感谢中国上海疾病预防控制中心提供一个中华按蚊菌群。 这项工作得到中国国家自然科学基金会(31301877)AX和由国家卫生研究院(Bethesda,MD,美国)授予(5R21AI094289)到IVS的支持。 AP和IVS 部分由关键技术和应用科学研究所(ICTAS)和NSF奖0850198支持。

参考文献

  1. Kamali,M.,Sharakhova,M.V.,Baricheva,E.,Karagodin,D.,Tu,Z.and Sharakhov,I.V。(2011)。 亚洲疟疾蚊子微卫星标记和反转断点的综合染色体图,

    。 J Hered 102(6):719-726。
  2. Kamali,M.,Xia,A.,Tu,Z.and Sharakhov,I.V。(2012)。 新的染色体系统发育支持冈比亚疟疾疟疾蚊子的矢量能力的重复起源复杂。 PLoS Pathog 8(10):e1002960。 
  3. Peery,A.,M.V.Sharakhova,Antonio-Nkondjio,C.,Ndo,C.,Weill,M.,Simard,F.,and I.V.Sharakhov。 2011. 改进用于研究非洲疟疾载体的群体遗传学工具箱 Anopheles nili :microsatellite mapping to chromosomes 。 寄生虫和向量 4:202.
  4. Sharakhova,M.V.,Xia,A.,Tu,Z.,Shouche,Y.S.,Unger,M.F。和Sharakhov,I.V。(2010)。 亚洲疟蚊的物理图,按蚊。 a> Am J Trop Med Hyg 83(5):1023-1027。 
  5. Sharakhova,M.V.,George,P.,Brusentsova,I.V.,Leman,S.C.,Bailey,J.A.,Smith,C.D。和Sharakhov,I.V。(2010)。 冈比亚按蚊异黄酮染色体的基因组图谱和表征。 BMC Genomics 11:459.
  6. Sharakhova,M.V.,Antonio-Nkondjio,C.,Xia,A.,Ndo,C.,Awono-Ambene,P.,Simard,F.and Sharakhov,I.V。 按蚊尼群岛的细胞遗传图谱:群体遗传学和比较物理图谱应用。 Infect Genet Evol 11(4):746-754。
  7. Xia,A.,Sharakhova,M.V.,Leman,S.C.,Tu,Z.,Bailey,J.A.,Smith,C.D。和Sharakhov,I.V。(2010)。 疟疾蚊子染色体的基因组景观和进化可塑性。<1>/em> 5(5):e10592 
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Xia, A., Peery, A., Kamali, M., Liang, J., Sharakhova, M. V. and Sharakhov, I. V. (2013). Fluorescence in situ Hybridization to the Polytene Chromosomes of Anopheles Mosquitoes. Bio-protocol 3(16): e860. DOI: 10.21769/BioProtoc.860.
  2. Kamali, M., Xia, A., Tu, Z. and Sharakhov, I. V. (2012). A new chromosomal phylogeny supports the repeated origin of vectorial capacity in malaria mosquitoes of the Anopheles gambiae complex. PLoS Pathog 8(10): e1002960.
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