generating dna probes to label the sex chromosomes of anopheles mosquitoes and preparing the hybridization solution

荧光 原位 杂交研究蚊子精子发生

Malaria imposes an enormous burden on the health and well-being of the global human population. In 2021, the World Health Organization (WHO) estimated that malaria caused 619,000 deaths, of which 96% occurred in Sub-Saharan Africa1. The disease is transmitted by mosquitoes belonging to the Anopheles genus, and in Sub-Saharan Africa, three species, namely Anopheles gambiae (An. gambiae), Anopheles coluzzi (An, coluzzi) and Anopheles arabiensis (An. Arabiensis) have a disproportionately large role in malaria transmission, accounting for 95% of malaria cases globally. Control programs relying on traditional methods such as insecticides and antimalarial drugs have saved millions of lives; however, in recent years, the rising resistance to these control methods has challenged their efficacy1,2. In addition, restrictions imposed by the COVID-19 pandemic have affected the availability of key malaria control interventions, which, according to the 2022 WHO World Malaria Report, has increased malaria incidence1. In the last two decades, novel genetic control methods have been developed in laboratory settings to target&#160;Anopheles mosquitoes3,4,5,6,7,8,9,10. Among these strategies, those based on gene drive systems (GDSs) and synthetic sex ratio distorters (SDs) seem promising. GDSs rely on the possibility of transmitting, at a very high frequency, a genetic modification that affects female fertility or impairs the parasite life cycle in the mosquito5,11,12. SDs, instead, act by skewing the sex ratio of a mosquito progeny toward males, which leads, over time, to the collapse of a target population due to a lack of females4,6,13. The main components of these genetic systems act primarily on the reproductive organs of the mosquitoes, where the gametes, eggs, and sperm are produced following meiotic division14.
In this protocol, advances in cytogenetic techniques are employed to explore spermatogenesis in An. gambiae focusing on the chromosomes&#39; behavior in situ. The structure of the mosquito testis and the biological processes that take place within it have been previously investigated using a number of cytological methods, such as immunofluorescence, fluorescent reporter transgenes, and DNA and RNA fluorescence in situ hybridization (FISH)15,16,17,18,19,20; the organs show a spindle-like shape, in which the lower pole is attached to a deferent duct connected to the male accessory glands. In the upper pole, the germline stem cells niche proliferates and differentiates into spermatogonia cells embedded inside spermatocysts formed by somatic cells. Following multiple rounds of mitotic division, the spermatogonia differentiate into spermatocytes, which enter meiosis. At prophase, autosome and sex chromosomes pair with their homologs, and crossing over takes place. After the meiotic divisions, round haploid spermatids are generated and enter spermiogenesis, and this process leads to the formation of mature haploid spermatozoa in which the cytoplasm has been removed, the nuclear chromatin is condensed, and flagella emerge at the basal part of the nuclei21,22 (Figure 1 and Figure 2).
In general, spermiogenesis starts around the mid-pupal stage, and mature spermatozoa can be detected in the late pupal stage in the sperm reservoir23. The maturation process of the spermatocysts continues during adult life23,24,25. In Anopheles testes, each step of spermatogenesis can be easily identified by looking at the nuclear morphology of the cells in each spermatocyst (Figure 2). Whole-mount fluorescence in situ hybridization (WFISH), described in this protocol, allows researchers to specifically label a chromosomal region and track it during spermatogenesis while preserving the native structure of the organ and cell nuclei position; this represents an advantage when compared to the standard DNA FISH protocol in which the organ is usually squashed, leading to tissue damage19. In the current protocol, fluorescent probes are used to stain repetitive sequences on the sex chromosomes and, thus, track their behavior during spermatogenesis, from diploid dividing cells to mature haploid spermatozoa. WFISH can be particularly useful for studying sex chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis4,19,26,27.
Given their role as malaria vectors, Anopheles mosquitoes are the target of an increasing number of genetic vector control strategies, which often act in the reproductive organs of these organisms. Several mosquito mutants and cytological phenotypes have been generated that require novel cytological techniques to be investigated26,27,28,29. The method described in this study sheds light on the understanding of spermatogenesis, as well as the cytological mechanisms behind genetic strategies that have the potential to control malaria-transmitting mosquitoes.

作者聚焦：全安装荧光原位杂交研究按蚊精子发生  

全安装荧光 原 位杂交研究 按 蚊精子发生

The scope of our research is to develop and share cost-effective and sustainable genetic strategies that aim at reducing the number of malaria-transmitting mosquitoes in South Saharan Africa. In the last few years, we have demonstrated the power of gene drive technologies in suppressing caged mosquito populations. We are currently running a number of experiments to challenge these technologies in more complex experimental setup and to answer the regulatory requirements that are absolutely necessary for the potential implementation of these technologies in the field.FISH is a molecular cytogenetic technique that allows for the staining of a specific DNA sequence. It typically involves the squashing of the organ of interest, which can results in the loss of information about the spatial arrangement of cells. Wellman FISH allows for the preservation of the native cytological structure of the organs.Wellman FISH can be used to study chromosome behavior in mosquito reproductive organs. Reproductive organs are the target of several genetic strategies for vector control. Wellmann FISH has the potential to shed light on the mechanism that take place in the reproductive organs of well-time mosquito, as well as in any genetic strain of interest.

Watch this Scientific Journal Video about Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution at JoVE.com

Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution  

生成DNA探针标记 按蚊 的性染色体并制备杂交溶液

首先使用商业提取试剂盒从蛹或成年雄性按蚊中提取基因组DNA。接下来，准备PCR反应混合物。通过进行PCR反应标记18 SR-DNA和卫星AGY 53B。为了制备杂交溶液，将5微升标记的DNA探针加入1.5毫升管中。然后加入五微升鲑鱼精子DNA。通过加入两体积的 100% 乙醇和 0.1 体积的三摩尔乙酸钠沉淀 DNA 探针。在零下 20 摄氏度下保持至少 2.5 小时。将试管以 17， 000 G 在 4 摄氏度下离心 20 分钟。然后除去乙醇，在室温下在黑暗中风干颗粒20分钟。接下来，在1.5毫升管中制备杂交缓冲液。涡旋溶液一分钟，让硫酸葡聚糖在 37 摄氏度下溶解 30 分钟。为了获得杂交溶液，将 DNA 调色板溶解在 20 至 30 微升的杂交缓冲液中。涡旋溶液一分钟。快速旋转，将试管存放在 37 摄氏度的黑暗中。

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JoVE Journal

Immunology and Infection

Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution

314 次观看.  Imperial College London. 首先使用商业提取试剂盒从蛹或成年雄性按蚊中提取基因组DNA。接下来，准备PCR反应混合物。通过进行PCR反应标记18 SR-DNA和卫星AGY 53B。为了制备杂交溶液，将5微升标记的DNA探针加入1.5毫升管中。然后加入五微升鲑鱼精子DNA。通过加入两体积的 100% 乙醇和 0.1 体积的三摩尔乙酸钠沉淀 DNA 探针。在零下 20 摄氏度下保持至少 2.5 小时。将试管以 17， 000 G 在 4 摄氏度下?...

视频: Generating DNA Probes to Label the Sex Chromosomes of Anopheles Mosquitoes and Preparing the Hybridization Solution  

Whole-Mount Fluorescence In Situ Hybridization to Study Spermatogenesis in the Anopheles Mosquito

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural changes to form haploid spermatozoa. Besides the biological aspect, studying spermatogenesis is of paramount importance for understanding and developing genetic technologies such as gene drive and synthetic sex ratio distorters, which, by altering Mendelian inheritance and the sperm sex ratio, respectively, could be used to control pest insect populations. These technologies have proven to be very promising in lab settings and could potentially be used to control wild populations of Anopheles mosquitoes, which are vectors of malaria. Due to the simplicity of the testis anatomy and their medical importance, Anopheles gambiae, a major malaria vector in sub-Saharan Africa, represents a good cytological model for studying spermatogenesis. This protocol describes how whole-mount fluorescence in situ hybridization (WFISH) can be used to study the dramatic changes in cell nuclear structure through spermatogenesis using fluorescent probes that specifically stain the X and Y chromosomes. FISH usually requires the disruption of the reproductive organs to expose mitotic or meiotic chromosomes and allow the staining of specific genomic regions with fluorescent probes. WFISH enables the preservation of the native cytological structure of the testis, coupled with a good level of signal detection from fluorescent probes targeting repetitive DNA sequences. This allows researchers to follow changes in the chromosomal behavior of cells undergoing meiosis along the structure of the organ, where each phase of the process can clearly be distinguished. This technique could be particularly useful for studying chromosome meiotic pairing and investigating the cytological phenotypes associated with, for example, synthetic sex ratio distorters, hybrid male sterility, and the knock-out of genes involved in spermatogenesis.

Given their simple anatomy, Anopheles testes offer a good cytological model for studying spermatogenesis. This protocol describes whole-mount fluorescence in situ hybridization, a technique used to investigate this biological process, as well as the phenotype of transgenic strains harboring mutations in the genes involved in sperm production.

Spermatogenesis is a complex biological process during which diploid cells undergo successive mitotic and meiotic division followed by large structural ...

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Begin by using a commercial extraction kit to extract the genomic DNA from the pupae or from adult male Anopheles mosquitoes. Next, prepare the PCR reaction mixture. Label the 18 SR-DNA and satellite AGY 53B by performing a PCR reaction.For the preparation of the hybridization solution, add five microliters of labeled DNA probe to a 1.5 milliliter tube. Then add five microliters of salmon sperm DNA. Precipitate the DNA probe by adding two volumes of 100%ethanol and 0.1 volume of three molar sodium acetate.Keep at minus 20 degrees Celsius for at least 2.5 hours. Centrifuge the tube at 17, 000 G for 20 minutes at four degrees Celsius. Then remove the ethanol and air dry the pellet at room temperature in the dark for 20 minutes.Next, prepare the hybridization buffer in a 1.5 milliliter tube. Vortex the solution for one minute and allow the dextran sulfate to dissolve at 37 degrees Celsius for 30 minutes. To obtain the hybridization solution, dissolve the DNA palette in 20 to 30 microliters of the hybridization buffer.Vortex the solution for one minute. Perform a quick spin, and store the tubes at 37 degrees Celsius in the dark.

Anopheles Mosquito Testis Dissection and Hybridisation

Anopheles Mosquito Testis Dissection and Hybridisation  

Begin by excising at least 20 testes from the pupae of Anopheles mosquitoes in sterile PBS. Transfer the testes to a clean microscope slide containing a fresh drop of PBS. Using P1000 boarded filtered tips or the tip of a dissection needle, transfer the testes into an embryo dish containing 3.7%formaldehyde in PBST.Incubate for 10 minutes at room temperature. Next, wash the testes in PBST for five minutes at room temperature. Transfer the testes to the embryo dish containing RNase A and incubated for a half hour at 37 degrees Celsius.Remove the RNA solution and add one milliliter of the penetrating solution. Incubate the testes in the penetrating solution at room temperature for 10 minutes. Then wash the testes and PBST two times for five minutes each.Transfer the testes to a 1.5 milliliter tube containing 20 to 30 microliters of hybridization solution with the labeled DNA probe. Incubate the mixture overnight at 37 degrees Celsius in the dark to allow hybridization. With the help of AP 1000 white bore filtered tip, transfer the testes into an embryo dish, then wash them in two XSSC for five minutes.Remove the SSC then mount the testes on a frosted glass slide using a commercially available mounting medium containing DAPI. Seal the slide with a cover slip sealant and incubate it at room temperature in the dark for two hours. A good level of hybridization allowed for the visualization of the targeted chromosomes throughout spermatogenesis.The pairing of the labeled sex chromosomes could be seen in the prebiotic and myotic stages. X or Y bearing spermatids could be followed throughout spermiogenesis marked by different levels of DNA condensation to the final step of arrow shaped mature spermatozoa.