桿狀病毒Cre/loxP 基因延長表現系統是一雙病毒系統。在過去此系統需要兩株桿狀病毒。一株攜帶被loxP序列包夾的外源基因表現匣，另一株則含有Cre重組酶表現匣。當兩株病毒同時進入目標哺乳動物細胞中，外源基因表現匣會被Cre重組酶由桿狀病毒基因體上切下，並重組成小環形DNA(episome)，此環形DNA可以穩定的存在細胞中，並長效表現外源基因。發展同時攜帶兩表現匣的單病毒Cre/loxP 基因延長表現系統，可使未來在應用時享有更多優點。然而，在生產病毒的過程中，Cre基因會滲漏導致外源基因嚴重缺失，因此為了在生產病毒過程中嚴格地限制Cre 的表現，我們利用專一性RNA剪接機制搭配特異性microRNA來抑制Cre基因於病毒生產流程中的滲漏。我們將四種不同的內含子插入Cre的開放閱讀框(open reading frame)，並篩選出在大腸桿菌以及昆蟲細胞中最能有效抑制Cre基因滲漏的內含子。此外為了更進一步的抑制Cre，我們在Cre 基因的3’未轉譯區(3’UTR)插入與MicroRNA bantam 完全互補的序列，透過這樣的策略，可減少Cre 成熟mRNA 99.8%的滲漏(相對於未調控組)，此新發展的Cre/loxP系統，在第二代病毒仍可保持93%外源基因，並在多種哺乳動物細胞中有高重組效率。為了證實本策略的泛用性，我們以同樣的方法，開發的單病毒載體的合成開關自切型CRISPR系統，相較於直接將Cas9表現匣與gRNA建構在一起的負對照組，新系統有更好的基因穩定度，第一代病毒含有89% 轉殖基因表現匣而對照組僅75%。在目標位點切割效率測試上，新系統的on target效率達20.4%，與過去共轉染質體所得的結果(23.7%)相近，但因新系統搭載病毒載體，未來可望遞送基因至難以轉染的細胞，更具應用潛力。

Cre/loxP‐based baculovirus(BV) system is a binary gene delivery system. In the past it required the delivery of two baculoviruses, one carrying the transgene cassette flanked by loxP and the other expressing Cre recombinase. Inside a target cell, transgene cassette is excised by the Cre recombinase and recircularizes to form an episomal DNA minicircle(episome). This minicircle can stably exist in target cell and enables long-term transgene expression. Developing a single BV system containing two cassettes may greatly benefit future applications. However, the leakage of cre may cause enormous loss of transgene in general BV production process. For stringently blocking Cre expression during the production of such single BV, we exploited the intron splicing mechanism in combination with specific microRNA to temporally control Cre expression. We tested 4 different introns, each of which was inserted into the cre open reading frame (ORF) and chose the best intron that successfully limited Cre expression in both E.coli and insect cell. To further downregulate Cre expression level, insect specific microRNA bantam complementary sequence was inserted into the 3’ untranslated region (UTR) of cre. These strategies altogether reduced approximately 99.8% of the mature Cre mRNA level in the insect cell comparing with the control group (BV with unmodified Cre expression cassette). This newly developed single Cre/loxP‐based baculovirus vector keeps 93% transgene expression cassette in the second passage, and maintains high recombinant efficiency in different types of mammalian cell.To confirm the applicability of our strategy, we used the same way to develop a single BV system of synthetic switch self-restrict CRISPR system .Comparing with the group which contains an unregulated Cas9 expression cassatte and gRNA cassatte (negative control), the BV genome stability of new single CRISPR system is better. This new single CRISPR system baculovirus keeps 89% transgene expression cassette and the negative control keeps only 75% in first passage. The on-target efficiency of new CRISPR system can achieve 20.4%. This result is similar to the result we got from previous transfection data (23.7%). Because our new single CRISPR system baculovirus can widely deliver gene into the cell which is difficult to transfect, new system will have more potential for gene editing.

摘要 I
Abstract II
誌謝 III
目錄 IV
圖表目錄 VII
第一章 文獻回顧 1
1-1桿狀病毒載體 1
1-2 桿狀病毒Cre/loxp基因延長表現系統 2
1-3 RNA剪接(RNA splicing) 4
1-4 MicroRNA簡介 4
1-4-1 MicroRNA bantam簡介 5
1-5 合成開關自切型CRISPR 系統 6
1-6研究動機 8
第二章 材料與方法 13
2-1 質體建構 13
2-1-1 建構重組酶質體(recombinase plasmid) 13
2-1-2 建構受質質體(substrate plasmid) 14
2-1-3 重組酶質體與受質質體共轉型實驗 15
2-1-4 建構MicoRNA bantam 完全互補序列 15
2-1-5 建構含重組酶與loxP序列的新重組酶系統質體 16
2-1-6 建構重組轉殖質體(recombinant donor plasmid) 17
2-2 製備重組桿狀病毒之相關技術 18
2-2-1 昆蟲細胞的培養與繼代 18
2-2-2 重組表現載體之轉置反應(transposition) 19
2-2-3 抽取重組Bacmid 20
2-2-4 轉染重組Bacmid至昆蟲細胞 20
2-2-5 重組桿狀病毒的生產放大與繼代 21
2-2-6 重組桿狀病毒之濃縮程序 21
2-2-7 桿狀病毒感染效價測定(infectious titer) 22
2-3基因重組桿狀病毒之轉導 22
2-3-1 哺乳動物細胞之培養 22
2-3-2 轉導哺乳動物細胞 23
2-4各種系統檢測分析 24
2-4-1 流式細胞儀分析(flow cytometry) 24
2-4-2即時偵測同步定量聚合酶連鎖反應(qPCR) 24
2-4-3反轉錄聚合酶連鎖反應(RT-PCR) 26
2-4-4即時偵測同步定量反轉錄聚合酶連鎖反應(qRT-PCR) 26
2-4-5西方點墨法分析 27
2-4-6 CRISPR 系統On-Target效率分析 28
2-4-7統計分析 29
第三章 實驗結果與討論 31
3-1 受intron調控的Cre基因在大腸桿菌中滲漏程度 31
3-1-1 CMV啟動子驅動的Cre基因在大腸桿菌中滲漏 31
3-1-2 rEF1-α啟動子驅動的Cre基因在大腸桿菌中滲漏 32
3-2 Cre 成熟mRNA在Sf9中表現量分析 33
3-2-1 以RT-PCR 進行Cre 成熟mRNA 定性分析 34
3-2-2 以qRT-PCR 進行Cre 成熟mRNA 定量分析 34
3-2-3 證實bantam 互補序列之效用 35
3-3 新Cre/loxp基因延長表現系統穩定度分析 36
3-4 哺乳動物細胞中重組效率分析 38
3-4-1 病毒劑量對於重組效率之影響 38
3-4-2 不同種類細胞內的重組效率分析 39
3-5 建構單病毒合成開關自切型CRISPR系統 40
3-5-1 比較經調控與未調控自切型CRISPR系統轉殖質體濃度 41
3-5-2自切型CRISPR系統在昆蟲細胞中的滲漏 41
3-5-3自切型CRISPR系統病毒基因體穩定度 42
3-5-4自切型CRISPR系統病毒on target 效率 42
3-6結論 44
第四章 未來展望 55
第五章 參考文獻 56

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