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標題: | 渦蟲基因遞送顯微注射器暨輔助之控溫平台研發 Microinjector and Temperature-Controlled Platform for Gene Delivery to Planarian |
作者: | Michael Lee 李林翰 |
指導教授: | 盧彥文(Yen-Wen Lu) |
關鍵字: | 基因遞送,顯微注射,渦蟲,再生, gene delivery,microinjection,planarian,regeneration, |
出版年 : | 2018 |
學位: | 碩士 |
摘要: | 本論文介紹一使用簡單材料製作之渦蟲基因遞送的微量注射系統。該系統包含了:(1)減少渦蟲運動的溫度控制平台以及(2)基因遞送微量注射器。
該系統中的溫度控制平台使用電阻測溫體量測平台溫度並配合微控制器和致冷晶片進行回饋控制以達到控制溫度的效果。採用不同的控制方法可以使平台達到較為快速、準確的溫度控制。基因遞送微量注射器則是使用電動缸(electric cylinder)為致動器並配合經熱熔拉伸成形的玻璃針來進行注射。連續注射實驗檢驗了注射器的穩定性。從嘗試拉針的結果得知在拉針的時候,(1)調整玻璃毛細管的溫度、(2)限制毛細管拉開的速度以及(3)施加不同大小的拉力,可以製作出不同外型和孔徑的玻璃針。 螢光微珠以及雙股核糖核酸注射實驗結果證實了本系統對於渦蟲基因遞送的適用性。利用本系統將DjAgo2雙股核糖核酸遞送進渦蟲身體裡將使渦蟲DjAgo2表現量下降並使渦蟲的再生功能缺失,此實驗成功的証明了本系統可用作餵食法的替代方案,除了能有效控制遺傳物質的注射量,還能用於研究需要飢餓條件之基因功能。 The thesis presents a microinjection system for gene delivery to planarian with simple materials. The system consists of two parts: a planarian-fixing platform and a nanoliter injector (nano-scale microinjector). The platform controls the temperature with the use of a microcontroller, a thermoelectric cooler, and a resistance thermometer. The microinjector is built with an electric cylinder and the glass needles are produced by pulling glass capillaries. The platform becomes more stable and accurate with better control methods. The processes and principles of pulling a needle with a micropipette puller are discussed: needles with desired shapes and orifice sizes can be produced with the puller by adjusting the temperature of a capillary, limiting the velocity of the capillary when it is being pulled and exerting a force to pull the capillary apart. The stability of the injector is confirmed by continuously injecting in the minimum volume. The adaptability of the system to planarian is validated by performing injections of fluorescent microbeads and dsRNA into planarian successfully. Delivery of DjAgo2 dsRNA leads to a reduction of DjAgo2 expression, which results in a degenerated phenotype of planarian. This validates our system as an alternative to ingestion, which performs delivery by feeding planarian genetic substances mixed food. Our system is applicable to studying the function of genes related to starvation condition. Besides, the dosage of genetic substances is consistent, which is required for the following analyses. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/69918 |
DOI: | 10.6342/NTU201800049 |
全文授權: | 有償授權 |
顯示於系所單位: | 生物機電工程學系 |
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