制备和检测胚胎显微操作用针的实用方法体系

Preparation and detection of pipettes for microinjection

目的介绍一种优化的制备及检测胚胎显微操作用针的实用方法体系。方法移卵管制作是用普通毛细管在酒精灯上拉制,小砂轮断针,针口用酒精灯火焰稍处理后备用;持卵针是将外径1.0mm的毛胚管在拉针仪上拉制,小砂轮断针,烧针仪上将开口处理成平滑内陷的马蹄形;注射针是将带内芯的外径1.0mm的特制毛胚管在拉针仪上拉制而成。注射针尖端充满注射溶液后,将注射针和持卵针连接到显微操作系统的操作臂上,先置于胚胎培养液内,用针尖轻轻碰触持卵针以破坏开口。之后转移到矿物油里,加压试注射,根据注射针开口液滴流出速度判断针尖是否通畅,流速是否合适。进一步用受精卵试注射进行验证。结果通过本方法体系制备的移卵管、持卵针和注射针均达到胚胎显微操作要求,解决了注射针口液体流速难以判断的问题。结论该胚胎显微操作用针的制备与检测方法体系使胚胎显微注射的前期工作得以标准化,提高了后续胚胎显微注射的效率。

Objective To introduce an optimized practical method of making and detecting pipettes for microinjection.Methods Transfer pipette was made from hard glass capillary. We softened the hard glass capillary by rotating it in a spirit-lamp flame,then moved out from the flame and quickly pulled it into two transfer pipettes.After broken by agrinding wheel,the tip of the pipette was fire-polished by quickly touching the flame to make a fine opening.A hard glass capillary（1.0mm,ouside diametre）was pulled into two holding pipettes by pipette Puller.The pipette shoulder was broken at desired position with a grinding wheel,then the fine pipette tip opening was heated by a microforge and shrinked into a diameter-15μm.Injection pipette could be made directly from a capillary with filament by Puller.The solution loaded injection pipette and holding pipette were assembled into the micromanipulator and could be checked before use.We transfered both pipettes into the zygotes media drop,touched the holding pipette with the tip of the injection pipette to make a ＂suitable＂opening.Then we switched injection pipette to the mineral oil and applied injection pressure through the injector to check whether the solution could come out of the tip smoothly and at a proper speed.It could be further verified by pronucleus microinjection of zygotes.Results The results showed that the method introduced in this paper could produce suitable pipettes for zygote microinjection.In particular,the method of detecting the opening of the injection pipette was helpful for achieving high efficiency of zygote microinjection.Conclusion The method introduced here to make and detect pipettes for microinjection is very helpful for establishing a standard microinjection manipulation procedure and improving the efficiency of zygote microinjection.