げっ歯類のマラリア原虫の遺伝的交雑の生産のためのプロトコル
Summary
げっ歯類のマラリア原虫の遺伝学的交配は蚊に2つの遺伝的に異なる寄生虫を供給することによって実行されます。組換え子孫は、蚊が感染したマウスを噛まないようにできるようにした後、マウスの血液からクローン化されています。このビデオでは、遺伝的交雑を生成する方法を示しています。<em>マラリアyoelii</em>と他のげっ歯類のマラリア原虫に適用可能である。
Abstract
抗マラリア薬への応答で、マラリア原虫の病原性の変化は、生物学的および医学的に重要である。リンケージマッピングは4-6 1-3およびヒトのげっ歯類のマラリア原虫の様々な特性の基礎となる遺伝子または遺伝子座の同定に成功につながっている。マラリア原虫yoeliiは野生のアフリカのげっ歯類から分離された多くのマラリアの種の一つであり、実験室で成長するように適応されています。この種は、人間のマラリア原虫の生物学的特性の多くを再現し、そのようなマイクロサテライトや増幅断片長多型（AFLP）マーカーなどの遺伝子マーカーはまた、寄生虫7月9日のために開発されている。従って、げっ歯類のマラリア原虫の遺伝学的研究は、 熱帯熱マラリア原虫の研究を補完するために実行することができます。ここで、我々は、P.の遺伝的交雑を生成するための手法を示します第一博士によって開拓されたyoelii。デビッドWalliker、リチャードカーター、そしてエディンバラ10の大学の同僚。
P.の遺伝的交雑yoeliiと他のげっ歯類のマラリア原虫は、関心の表現型と蚊は4日、感染後に感染したマウスを餌にできるため、異なる2つの遺伝的に異なるクローンのgametocytesを含む接種でマウスのハツカネズミを感染させることによって行われます。マウスの血液中の男性と女性gametocytesの有無を顕微鏡供給する前に確認されている。授乳後48時間以内に、蚊の中腸では、半数体gametocytesは、男性と女性の配偶子に分化受精させる、と二倍体の接合子（図1）を形成する。オーキネートへの受精卵の開発中に、減数分裂は11を発生することが表示されます。受精卵は、2つの遺伝的に異なる寄生虫、遺伝的交流（。相同染色体のペアの非姉妹染色分体の間に染色体の再集合し、クロスオーバー、図2）の配偶子間のクロス受精によって派生している場合は、組換えの結果、発生する可能性があります。相同遺伝子座の遺伝物質の。それぞれの接合子は、4倍体の核につながる、二つの連続核分裂を起こす。オーキネートはさらにオーシストに開発しています。オーシストが成熟したら、スポロゾイト（クロスの子孫）の数千人が形成され、蚊のhemocealに放出されています。スポロゾイトが唾液腺から採取し、前赤血球と赤血球ステージの開発が行われる新しいマウス宿主に注入されています。赤血球の形態はクローニングと遺伝子連鎖地図作成の前に親の行を区別する文字に関して分類されます。個々の親のクローンの制御の感染症は、遺伝的交雑の生産と同じ方法で実行されます。
Protocol
Discussion
我々はまた、他のげっ歯類malariasの遺伝的交雑の製造に適用可能である齧歯類マラリア原虫yoelii、の遺伝的交雑の生産のための手法を示します。単一の親のクローンを持つマウスの感染は通常、親がクロスを実行する前に、機能的な配偶子の生産で有能であることを保証するために親の寄生虫の転送に成功したことを判断するために実行されます。
蚊を介して転送…
Divulgations
The authors have nothing to disclose.
Acknowledgements
私たちは、原稿の重要な読書のために、博士ランディエルキンズ、ロビンKastenmayer、テッドトーリー、ダンパレとToviリーマンに感謝。この作品は、＃2007CB513103、学内研究の部門の学内研究プログラム、アレルギーや国立感染症研究所、国立衛生研究所によって、中国の973国家基礎研究プログラムによってサポートされていました。我々は援助のためにNIAID学内エディタブレンダレイマーシャルに感謝。
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.
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