生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Summary

啮齿类动物疟疾寄生虫的基因杂交进行喂养蚊子的两个基因的不同的寄生虫。克隆小鼠血液，让蚊虫叮咬感染小鼠后，重组的后代。本视频演示了如何产生遗传杂交<em>约氏疟原虫</em>，并适用于其他啮齿类动物疟疾寄生虫。

Abstract

在抗疟疾药物，疟疾寄生虫的致病性变异是生物和医疗的重要性。连锁图谱已经导致成功的基本在^1-3啮齿类动物和人类^4-6疟疾寄生虫的各种性状的基因或位点的识别。 氏疟原虫疟疾寄生虫是许多疟疾从非洲野生啮齿类动物中分离的物种之一，已经适应了在实验室中生长。该物种繁殖许多人类疟疾寄生虫的生物学特性，遗传标记，如微和扩增片段长度多态性（AFLP）标记也已开发的^寄生虫 7-9 。因此，在啮齿类动物疟疾寄生虫的基因研究可以进行补充对恶性疟原虫的研究。在这里，我们展示的技术， 生产 P.遗传交叉约氏疟原虫 ，第一个率先由Drs。大卫Walliker，理查德卡特和他的^同事在10爱丁堡大学。

在体育的遗传杂交约氏疟原虫和其他啮齿类动物疟疾寄生虫是由一个包含了两个基因不同的克隆，在利益的表型，使蚊子感染小鼠感染后第4天的饲料不同配子体接种感染小鼠的小家鼠。小鼠血液中的男性和女性配子体的存在是微观喂奶前确认。喂奶后48小时内，在蚊子的中肠，单倍体的配子体分化为雌雄配子，施肥，并形成一个二倍体的合子（图1）。在发展成一个动合子受精卵，减数分裂发生^11。如果通过基因的两个不同的寄生虫病，遗传交流（染色体重组和跨接一对同源染色体的非姐妹染色单体之间;图2）配子之间的相互交流中衍生的受精卵是可能发生的，在重组同源基因位点的遗传物质。每个受精卵经过连续两次核部门，导致4个单倍体核。一个动合子，进一步发展成一个卵囊。一旦卵囊的成熟，成千上万的孢子（跨后代）形成和释放到蚊子hemoceal。孢子是收获的唾液腺发生前红细胞和红细胞阶段发展到一个新的小鼠主机，的注入。红细胞形式的克隆和分类方面区分亲本遗传连锁映射之前的字符。个别家长克隆控制感染是在相同的方式作为一种遗传性的交叉的生产。

Protocol

无菌技术必须适用于所有材料将动物给药，以避免无意中引入到小鼠体内，可以混淆实验结果外源性感染制剂。 1。血液阶段的疟疾寄生虫感染的实验室小鼠在室温下解冻，需要跨越两个小瓶冷冻血液阶段疟疾寄生虫。在这个例子中，两个鼠疟原虫株氏疟原虫约氏疟原虫和氏疟原虫nigeriensis。 配合一个22-30号（G）的针头注射器，并绘制了400μL无菌?…

Discussion

我们展示的技术，生产的灭鼠疟疾氏疟原虫，这也适用于其他啮齿类动物malarias的遗传杂交生产中的遗传交叉。单身父母克隆小鼠的感染通常以确定父母寄生虫的成功传输，以确保父母在生产前进行跨功能的配子主管。

成功传输通过蚊子是由多种因素，包括温度insectary，用于灭鼠疟疾寄生虫的种类，和血阶段寄生虫的剂量​​（接种量）的影响。虽然体育疟原虫…

Materials

Material Name	Type	Company	Catalogue Number	Comment
Glyerolyte 57 solution		Cenmed	4A7833
Mouse Mus musculus		Charles River Laboratory		Female, inbred, strain Balb/C
Heat-inactivated calf serum		Invitrogen	26010-066
Phosphate buffered saline (PBS) solution		Invitrogen	10010-072	pH 7.4; Cell Culture grade
Malaria parasite Plasmodium yoelii yoelii 17XNL(1.1)		MR4	MRA-593	deposited by DJ Carucci
Malaria parasite Plasmodium yoelii nigeriensis N67		MR4	MRA-427	deposited by W Peters, BL Robinson, R Killick Kendrick
Mosquito Anopheles stephensi		MR4	MRA-128	deposited by MQ Benedict
Cellometer automatic cell counter		Nexcelom Biosciences	Cellometer Auto T4
Cellometer CP2 disposable hemacytometer		Nexcelom Biosciences	Cellometer CP2
High Pure PCR template preparation kit		Roche Applied Science	11 796 828 001
Calcium chloride		Sigma-Aldrich	C5670	Cell culture tested; insect cell culture tested
Giemsa stain, modified		Sigma-Aldrich	GS500
Ketamine hydrochloride		Fort Dodge Animal Health	NDC 0856-2013-01	Pharmaceutical grade; concentration to 100 mg/mL
Potassium chloride		Sigma-Aldrich	P5405	Cell culture tested; insect cell culture tested
Sodium chloride		Sigma-Aldrich	S5886	Cell culture tested; insect cell culture tested
Trisodium citrate dihydrate		Sigma-Aldrich	S4641
Xylazine		Akorn Inc.	4811-20ml	Pharmaceutical grade; concentration to 20 mg/mL
Glass wool		VWR	32848-003
Glass capillary (1 μL)		VWR	53440-001
Hemocytometer		VWR	15170-168	Complete chamber set
Homogenizer		VWR	KT749520-0090	Pestle with matching tube, 1.5 mL

SUPPLEMENTARY MATERIALS:

• Maintenance of laboratory mice
• Maintenance of laboratory mosquitoes
• Microscopic examination of thin blood smears stained with Giemsa stain
• Measurement of red blood cell density

Maintenance of laboratory mice

Females of inbred laboratory mouse strain BALB/c, aged 5 to 8 weeks old, are used in the study. Mice are housed in a standard solid-bottom polycarbonate cage with wire-bar lid, equipped with feeder and a water bottle. Mice are maintained at a constant temperature (25 ± 1°C) on 12:12 hour light:dark cycle. Mice are allowed to feed on 2018S Harlan Teklad Global 19% protein extruded rodent diet (sterilizable; from Harlan-Teklad) and supplied with acidified drinking water ad libitum. Experiments on animals are performed in accordance with the guidelines and regulations set forth by the Animal Care and Use Committee at the National Institute of Allergy and Infectious Disease under protocol LMVR11E (National Institutes of Health, Bethesda, Maryland).

Maintenance of laboratory mosquitoes

Mosquitoes are from a laboratory-bred colony of Anopheles stephensi. The adults are maintained in nylon cages kept in a temperature- and humidity-controlled room (23 to 25°C for Plasmodium yoelii and Plasmodium chabaudi, and 19 to 21°C for Plasmodium berghei; 80 to 95% humidity; on 12:12 hours light:dark cycle). Adult mosquitoes are fed with 10% glucose and 2.00% para-aminobenzoic acid (PABA) supplemented water solution. To obtain high-quality adults, 500 larvae are grown in a low-density condition in 1 L of distilled water in a 1,000-cm³ open dish supplied with approximately 1 mg of sodium bicarbonate. After hatching, the larvae are given tetramin powder (PETCO) until they develop into the pupa stage and are transferred to the adult mosquito cages for emerging.

Microscopic examination of thin blood smears stained with Giemsa stain

Using clean scissors snip off the tip (1.0 mm) of the infected mouse’s tail. Place one drop (0.5-1.0 μL) of tail blood onto a clean specimen slide. Mouse will stop bleeding in 1-2 min. Place a clean spreader slide on top of the blood drop, maintaining it at a 45° angle relative to the specimen slide, and allow the blood to adsorb to the entire width of the spreader. Hold the specimen slide and push forward the spreader slide rapidly and smoothly to produce a thin smear. Let the blood film dry, and then immerse the slides in absolute methanol. Allow the slide to air dry once more before covering it with Giemsa stain (10% Giemsa dye in distilled water). After incubating the thin blood films for 10-15 min at room temperature, carefully rinse the slides with tap water and let it air dry. Examine the number of infected red blood cells (iRBC; see Figure 3 for morphology of infected RBC) under a light microscope with immersion oil at 1000x magnification (with 100x objective lens) and calculate parasitemia (the number of iRBC per 100 RBC counted). Different strains of malaria parasites vary in growth rate and pathogenicity. Monitoring of blood stage parasitaemias can be performed 24hrs after injections, depending on the dose of the blood stage malaria parasites. For example, mice will be microscopically positive 24 hrs when injected with 10⁷ infected RBC intraperitoneally or 10⁶ infected RBC intravenously.

Measurement of red blood cell density

Like the levels of parasitaemias, red blood cell (RBC) density in infected mice varies throughout the course of infection. RBC density should be measured within 1-2 hrs before the start of the single- and mixed-clone infection and the cloning experiments. There are two methods for measurement of RBC density: a manual counting using Neubauer hemocytometer and an automatic counting using a Cellometer (Nexcelom Bioscience). In both methods, withdraw 1 μL of mouse tail blood using a glass capillary (VWR) and dilute in 10 mL of PBS and mix well. To use a Neubauer hemocytometer, load 20 μL of the suspension onto the hemacytometer. Place the hemacytometer on a light microscope with 10x objective lens. The hemacytometer contains a grid divided into 9 large squares, and 4 large squares at the corner are further divided into 16 small squares. Count the total number of cells in each of the 16 small squares in the four corner squares. To avoid counting bias or counting cells that overlap a grid line, count a cell as “in” if it overlaps the top or right lines and “out” if it overlaps the bottom or left lines. Estimate the number of cells per one small square and divide by 0.00625 (the volume of one small square is 6.25 nL). This yields the number of cells per microliter (μL). From this data, calculate the final red blood cell density by multiplying with 10,000 (a dilution factor). Rinse the cover slip and counting chamber with distilled water and 70% ethanol; air dry. Alternatively, load 20 μL of the suspension onto a Cellometer counting chamber slide. Insert the slide into a Cellometer slide chamber (the reader). Start the Cellometer software, select the “red blood cell” option, and enter a dilution factor of 10,000. Record the RBC density.