Summary

通过显微注射在类 圆线虫 物种中产生转基因和敲除

Published: October 07, 2021
doi:

Summary

寄生线虫 类圆线 虫粪类和大 鼠类圆线 虫的功能基因组工具包包括转基因、CRISPR/Cas9 介导的诱变和 RNAi。该协议将演示如何使用性腺内显微注射将转基因和CRISPR成分引入 粪类猪笼草鼠李糖杆菌中。

Abstract

类圆线虫属由多种具有不同宿主范围的穿透皮肤线虫组成,包括粪类线虫和大鼠类圆线虫S. stercoralis是一种人类寄生虫,穿透皮肤的线虫,感染约6.1亿人,而大鼠寄生虫S. ratti粪类S. stercoralis密切相关,通常用作粪类菌丝虫的实验室模型。S. stercoralisS. ratti都很容易通过性内显微注射的外源核酸递送技术产生转基因和敲除,因此,已经成为其他尚未适应该技术的寄生蠕虫的模型系统。

寄生 类圆线虫 成虫栖息在宿主的小肠中,并通过粪便将后代释放到环境中。一旦进入环境,幼虫就会发育成自由生活的成虫,它们生活在粪便中并产生必须找到并入侵新宿主的后代。这种环境生成是 类圆线虫 物种所独有的,在形态上与自由生活线虫 秀丽隐杆线虫 模型足够相似,为 秀丽隐杆线虫 开发的技术可以适应与这些寄生线虫一起使用,包括性腺内显微注射。使用性腺内显微注射,可以将各种各样的转基因引入类 圆线虫中。CRISPR / Cas9组分也可以显微注射以产生突变 的圆线虫 幼虫。这里,描述了性腺内显微注射到 类圆线虫的技术, 包括自由生活成人的制备,注射程序和转基因后代的选择。包括使用CRISPR / Cas9诱变产生的转基因 类圆线虫 幼虫的图像。本文的目的是使其他研究人员能够使用显微注射来产生转基因和突变 的类圆线虫

Introduction

与更广泛认可的钩虫和蛔虫虫相比,类粪类圆线虫长期以来一直被忽视为一种重要的人类病原体以前对蠕虫负担的研究往往严重低估了粪类链球菌的患病率,因为粪类疟原虫的常见诊断方法的敏感性较低 2.近年来,基于改进的诊断工具的流行病学研究估计,粪类菌感染的真实患病率远高于先前报告的,全世界约有6.1亿人2

S. stercoralis和其他类圆线虫物种,包括密切相关的大鼠寄生虫和常见的实验室模型S. ratti,都有一个不寻常的生命周期,对实验基因组研究有利,因为它由寄生和自由生活(环境)第3代组成(图1)。具体来说,S. stercoralisS. ratti都可以通过单一的自由生活世代循环。自由生活的一代由后寄生虫幼虫组成,这些幼虫发育成自由生活的成年雄性和雌性;自由生活的成虫的所有后代都发育成感染性幼虫,必须感染宿主才能继续生命周期。此外,这种环境或自由生活的一代可以在实验室中通过实验操纵。由于自由生活的类圆线虫成虫和秀丽隐杆线虫成虫具有相似的形态,因此最初为秀丽隐杆线虫开发的性腺内显微注射等技术可以适用于自由生活的成年类圆线虫45。虽然DNA通常被引入自由生活的成年雌性中,但类圆线虫的雄性和雌性都可以被显微注射6。因此,功能基因组工具可用于询问类圆线虫生物学的许多方面。其他寄生线虫缺乏自由生活的一代,因此,不容易适应功能基因组技术3

Figure 1
图1:类圆线虫的粪类动物生命周期。 粪类孢子虫寄生雌性栖息在其哺乳动物宿主(人类,非人类灵长类动物,狗)的小肠中。寄生雌性通过单性生殖繁殖,并在小肠内产卵。卵在宿主体内孵化成后寄生幼虫,然后与粪便一起进入环境。如果寄生后幼虫是雄性,它们会发育成自由生活的成年雄性。如果寄生后幼虫是雌性,它们可以发育成自由生活的成年雌性(间接发育)或第三阶段感染性幼虫(iL3s;直接发育)。自由生活的雄性和雌性有性繁殖以产生被限制成为iL3的后代。在某些情况下,粪类杀伤性链球菌也可以进行自身感染,其中一些后寄生幼虫留在宿主肠道内,而不是通过粪便进入环境。这些幼虫可以在宿主体内发育成自身感染性幼虫(L3a),穿透肠壁,通过身体迁移,最终返回肠道成为生殖成虫。S. ratti的生命周期是相似的,除了S. ratti感染大鼠并且没有自身感染周期。环境生成是使用类圆线虫物种进行遗传研究的关键。自由生活的成年雌性(P0)可以显微注射;它们的后代,全部将成为iL3s,是潜在的F1转基因。这个数字是从Castelletto等人修改而来的。3. 请按此浏览此图的大图。

S. stercoralis 与其他胃肠道人寄生虫线虫共享其生物学的许多方面,包括宿主入侵和宿主免疫调节。例如, Necator 属和 Ancylostoma 属的人寄生虫钩虫也通过皮肤渗透感染,以类似的方式穿过身体,最终作为寄生成虫居住在小肠7中。因此,许多胃肠道线虫可能使用常见的感觉行为和免疫规避技术。因此,从 类圆线虫 中收集的知识将补充其他遗传上不太易处理的线虫的发现,并导致对这些复杂而重要的寄生虫有更完整的了解。

该显微注射方案概述了将DNA引入圆 线虫 自由生活成年雌性以制造转基因和突变后代的方法。描述了菌株维持要求,包括用于显微注射的成虫的发育时间和转基因后代的收集。包括实验方案和完整的显微注射技术演示,以及培养和筛选转基因后代的方案,以及所有必要设备和消耗品的清单。

Protocol

注意:沙鼠用于通过 S. stercoralis,大鼠用于通过 S. ratti。所有程序均由加州大学洛杉矶分校动物研究监督办公室(协议编号2011-060-21A)批准,该办公室符合AAALAC标准和实验室动物护理和使用指南。以下任务必须在显微注射前至少一天完成:蠕虫培养,制备显微注射垫,为显微注射混合物创建结构,以及将细菌(大肠杆菌 HB101)扩散到6厘米线虫生长培养基(NGM)平板<sup class="xre…

Representative Results

如果实验成功,F1 幼虫将表达感兴趣的转基因和/或突变表型(图4)。然而,转化率变化很大,取决于结构,蠕虫的健康状况,注射后培养条件和实验者的技能。一般来说,成功的实验将产生每只注射雌性>15 F 1 幼虫,荧光标记物的转化率为>3%。如果活的后代总数平均少于10只幼虫/雌性,那么该结构可能是有毒的,并且转化的幼虫无法存活。发现大量荧光卵?…

Discussion

该显微注射方案详细介绍了将转基因结构和CRISPR / Cas9介导的诱变引入 粪类链球菌鼠李糖杆菌的方法。 对于粪 类葡萄球菌鼠李松,注射后存活率和转基因或诱变率受制于几个可以微调的变量。

成功转基因的第一个关键考虑因素是如何构建质粒转基因。以前的研究发现,类 圆线虫 中外源转基因的表达需要使用 类圆线虫 5’启动子…

Declarações

The authors have nothing to disclose.

Acknowledgements

pPV540和pPV402是宾夕法尼亚大学James Lok博士的善意礼物。我们感谢阿斯特拉·布莱恩特对手稿的有益评论。这项工作由Burroughs-Wellcome Fund Researchs in the Pathogenesis of Disease Award,Howard Hughes Medical Institute教师学者奖和美国国立卫生研究院R01 DC017959(E.A.H.)资助。

Materials

(−)-Nicotine, ≥99% (GC), liquid Sigma-Aldrich N3876-5ML nicotine for paralyzing worms
3" iron C-clamp, 3" x 2" (capacity x depth) VWR 470121-790 C-clamp to secure setup to bench top
Agarose LE Phenix RBA-500 agarose for slides
Bone char, 4 lb pail, 10 x 28 mesh Ebonex n/a charcoal for fecal-charcoal cultures
Bone char, granules, 10 x 28 mesh Reade bonechar10x28 charcoal for fecal-cultures (alternative to the above)
Coarse micromanipulator Narishige MMN-1 coarse micromanipulator
Corning Costar Spin-X centrifuge tube filters Fisher 07-200-385 microfilter column
Cover glass, 48 x 60 mm, No. 1 thickness Brain Research Lab 4860-1 coverslips (48 x 60 mm)
Deep Petri dishes, heavy version with 6 vents, 100 mm diameter VWR 82050-918 10 cm Petri dishes (for fecal-charcoal cultures)
Eisco retort base w/ rod Fisher 12-000-101 stand for Baermann apparatus
Eppendorf FemtoJet microinjector microloader tips VWR 89009-310 for filling microinjection needles
Fisherbrand absorbent underpads Fisher 14-206-62 bench paper (for prepping)
Fisherbrand Cast-Iron Rings Fisher 14-050CQ Baermann o-ring
Fisherbrand tri-cornered polypropylene beakers Fisher 14-955-111F Plastic beaker (for mixing)
Fisherbrand tri-cornered polypropylene beakers Fisher 14-955-111F Plastic beaker (for catch bucket/water bucket)
Fisherbrand tri-cornered polypropylene beakers Fisher 14-955-111F Plastic beaker (x2) (to make holder)
Gorilla epoxie in syringe McMaster-Carr 7541A51 glue (to attach tubing)
Halocarbon oil 700 Sigma-Aldrich H8898-50ML halocarbon oil
High-temperature silicone rubber tubing for food and beverage, 1/2" ID, 5/8" OD McMaster-Carr 3038K24 tubing (for funnel)
KIMAX funnels, long stem, 60° Angle, Kimble Chase VWR 89001-414 Baermann funnel
Kimberly-Clark Professional Kimtech Science benchtop protectors Fisher 15-235-101 bench paper (for prepping)
Leica stereomicroscope with fluorescence Leica M165 FC GFP stereomicroscope for identifying and sorting transgenic worms
microINJECTOR brass straight arm needle-holder Tritech MINJ-4 microinjection needle holder
microINJECTOR system Tritech MINJ-1 microinjection system
Mongolian Gerbils Charles River Laboratories 213-Mongolian Gerbil gerbils for maintenance of S. stercoralis, male 4-6 weeks
Nasco Whirl-Pak easy-to-close bags, 18 oz VWR 11216-776 Whirl-Pak sample bags
Nylon tulle (mesh) Jo-Ann Fabrics zprd_14061949a nylon mesh for Baermann holder
Platinum wire, 36 Gauge, per inch Thomas Scientific 1233S72 platinum/iridium wire for worm picks
Puritan tongue depressors, 152 mm (L) x 17.5 mm (W) VWR 62505-007 wood sticks (for mixing samples)
QIAprep Spin Miniprep Kit (250) QIAGEN 27106 QIAGEN miniprep kit
Rats-Long Evans Envigo 140 HsdBlu:LE Long Evans rats for maintenance of S. ratti, female 4-6 weeks
Rats-Sprague Dawley Envigo 002 Hsd:Sprague Dawley SD rats for maintenance of S. ratti, female 4-6 weeks
Really Useful Boxes translucent storage boxes with lids, 1.6 L capacity, 7-5/8" x 5-5/16" x 4-5/16" Office Depot 452369 plastic boxes for humidified chamber
Shepherd techboard, 8 x 16.5 inches Newco 999589 techboard
Stainless steel raised wire floor Ancare R20SSRWF wire cage bottoms
StalkMarket compostable cutlery spoons, 6", white, pack of 1,000 Office Depot 9587303 spoons
Stender dish, stacking type, 37 x 25 mm Carolina (Science) 741012 watch glasses (small, round)
Stereomicroscope Motic K-400 LED dissecting prep scope
Storage tote, color clear/white, outside height 4-7/8 in, outside length 13-5/8 in, Sterilite Grainger 53GN16 plastic boxes for humidified chamber
Sutter P-30 micropipette puller Sutter P-30/P needle puller with platinum/iridium filament
Syracuse watch glasses Fisher S34826 watch glasses (large, round)
Thermo Scientific Castaloy fixed-angle clamps Fisher 05-769-2Q funnel clamps (2x)
Three-axis hanging joystick oil hydrolic micromanipulator Narishige MM0-4 fine micromanipulator
United Mohr pinchcock clamps Fisher S99422 Pinch clamps (2x)
Vented, sharp-edge Petri dishes (60 mm diameter) Tritech Research T3308P 6 cm Petri dishes (for small-scale fecal-charcoal cultures)
VWR light-duty tissue wipers VWR 82003-820 lining for Baermann holder
watch glass, square, 1-5/8 in Carolina (Science) 742300 watch glasses (small, square)
Whatman qualitative grade plain circles, grade 1, 5.5 cm diameter Fisher 09-805B filter paper (for 6 cm Petri dishes)
Whatman qualitative grade plain circles, grade 1, 9 cm diameter Fisher 09-805D filter paper (for 10 cm Petri dishes)
World Precision Instrument borosilicate glass capillary, 1.2 mm x 4 in Fisher 50-821-813 glass capillaries for microinjection needles
X-Acto Knives, No. 1 Knife With No. 11 Blade Office Depot 238816 X-Acto knives without blades to hold worm picks
Zeiss AxioObserver A1 Zeiss n/a inverted microscope

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Castelletto, M. L., Hallem, E. A. Generating Transgenics and Knockouts in Strongyloides Species by Microinjection. J. Vis. Exp. (176), e63023, doi:10.3791/63023 (2021).

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