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研究生:洪翎甄
研究生(外文):Ling-Zhen Hong
論文名稱:運用CRISPR / Cas9基因編輯技術產生nrz斑馬鱼突變體並解析其特性
論文名稱(外文):Characterization of nrz zebrafish mutants generated by CRISPR/Cas9
指導教授:李士傑李士傑引用關係
指導教授(外文):Shyh-Jye Lee
口試委員:吳益群賴時磊
口試委員(外文):Yi-Chun WuShih-Lei Lai
口試日期:2019-07-23
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:分子與細胞生物學研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2019
畢業學年度:107
語文別:中文
論文頁數:58
中文關鍵詞:nrz外包斑馬魚肌動蛋白/肌球蛋白環卵黃融合細胞層
DOI:10.6342/NTU201902466
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Nrz屬於Bcl-2蛋白質家族,負責調控細胞凋亡。在哺乳類細胞中,Nrz做為抗細胞凋亡因子,抑制因生長因子剝奪或是促細胞凋亡因子增加引起的細胞凋亡。 Nrz同時也做為鈣離子(Ca2+)調控因子,與位於內質網的肌醇三磷酸受器(IP3R, inositol 1,4,5-trisphosphate receptor)結合,抑制鈣離子通過肌醇三磷酸受器從內質網釋放至胞漿(cytosol)。在斑馬魚胚胎早期發育的外包階段(epiboly stage),Nrz表達於卵黃融合細胞層(Yolk syncytial layer, YSL)的內質網與粒線體胞器。透過嗎啉基(morpholino)對Nrz基因進行基因敲落(Knockdown),導致胚胎的外包停滯(epiboly arrest),外包動作停止後胚盤(blastoderm)邊緣快速收縮,最後使胚盤脫離卵黃細胞,造成胚胎死亡。外包停滯缺陷並非因為失去Nrz抑制細胞凋亡功能,而是因為失去Nrz抑制鈣離子釋放功能造成。在Nrz基因敲落的斑馬魚胚胎中,卵黃融合細胞層的胞漿鈣離子濃度上升,鈣離子進而刺激鈣調蛋白/肌球蛋白輕鏈激酶(calmodulin/MLCK)的訊息傳遞路徑,因此使提供收縮力量的肌動蛋白/肌球蛋白環(actomyosin-ring)提早形成,導致胚盤邊緣收縮。然而上述研究都是根據嗎啉基誘導的基因敲落所提出,為了能夠更進一步探討證實和探討Nrz在斑馬魚胚胎中的功能,我們透過CRISPR/Cas 9基因修改技術剃除(knockout)Nrz基因,產生Nrz突變魚(mutants)。合子(zygotic)與母源(maternal)nrz突變胚胎沒有表現出大量的外包缺陷,但是母源合子突變胚胎(maternal-zygotic embryos)出現嚴重的外包缺陷。另外,胚胎邊緣收縮缺陷不再局限於原腸胚期(gastrulation period),還出現在更早的囊胚期(blastula period),同時在表現缺陷的胚胎也觀測到肌動蛋白環的提早形成。我們利用基因修改技術證實Nrz在原腸胚期的功能,透過Nrz突變體可以更進一步釐清早期胚胎發育的外包調控機制,甚至是後來的器官生成機制,而這是嗎啉基誘導的基因敲落無法做到的。
Nrz is a Bcl-2 family protein that is known as an apoptosis regulator. It can inhibit apoptosis induced by growth factor deprivation or other pro-apoptotic factors in mammalian cell lines. In addition, Nrz has also been shown to inhibit IP3-induced Ca2+ release during embryogenesis in zebrafish. It expresses in the external yolk syncytial layer. Knockdown of Nrz with morpholino (MO) leads to epiboly arrest, blastoderm margin constriction and the detachment of blastoderm from yolk cell. However, the epiboly defect is due to the dysregulation of Ca2+ homeostasis, instead of Caspase-mediated apoptosis. Loss of Nrz results in the increase of cytosol Ca2+ amplitude. Ca2+ activates calmodulin/MLCK pathway and subsequently premature formation of the actin-myosin ring in the E-YSL. All of the above information are from MO-based studies, while concerns have been raised for the potential non-specificity and secondary effects induced by MO. To further confirm and clarify the functions of Nrz in zebrafish embryogenesis, here I have identified several nrz mutants-generated by CRISPR/Cas9 technology. I found that zygotic and maternal nrz mutants did not exhibit severe epiboly defects as shown in the nrz-MO studies. Those homozygous zygotic nrz mutants could be raised to adulthood to produce the F3 maternal-zygotic (MZ) mutants. Interesting, those MZ nrz mutants showed severe premature blastoderm constriction defect and premature actin-ring formation in YSL region between the blastula and gastrula period. More importantly those defects could be significantly rescued by injecting wild-type nrz mRNA. In conclusion, I validated the functions of Nrz during gastrulation in nrz mutants. The availability of nrz mutants enables us to further elucidate the regulatory mechanisms during both embryogenesis and organogenesis, which will complement the MO-based knockdown approaches.
中文摘要 II
Abstract IV
Acknowledgement 致謝 V
Contents VII
Introduction 1
Materials and methods 5
Zebrafish maintenance and embryo culture 5
Generation of CRISPR/Cas9 mediated nrz knockout zebrafish 5
Polymerase chain reaction and Mly I restriction digestion 7
RNA isolation and reverse transcription-PCR (RT-PCR) 7
Zebrafish nrz cloning and expression vector construction 8
mRNA in vitro transcription 8
Microinjection 9
Extraction mitochondria and Immunoblotting 9
Rhodamine-phalloidin staining 10
Results 11
Generation of nrz mutants 11
Epiboly arrest occurs in nrz mutant embryos 14
Rescue of maternal-zygotic nrz embryos 17
Actin-ring formation in the YSL 18
Discussion 20
References 23
Tables 28
Table 1. Guide RNAs in this study. 28
Table 2. Priners in this study. 29
Table 3. Sequences of F1 nrz mutants. 30
Table 4. Sequences of nrz mutants. 31
Figures 32
Figure 1. Nrz genomic structure and target sites of CRISPR guide RNAs. 32
Figure 2. Activity test of guide RNAs. 34
Figure 3. Generation nrz zygotic mutants. 35
Figure 4. Restriction digestion analysis of mutant alleles. 36
Figure 5. Generation nrz maternal-zygotic mutants. 37
Figure 6. Partial sequencing chromatogams of wildtype and nrz mutant alleles. 38
Figure 7. Predicted Nrz functional domain changes for nrz mutant alleles. 39
Figure 8. Nrz protein in nrz mutant alleles. 41
Figure 9. F2 progenies of nrz mutant alleles have a high survival rate and few abnormality. 42
Figure 10. Genotyping of abnormal nrz F2 progenies from heterozygous mutants incross. 43
Figure 11. Maternal and zygotic effects of nrz on embryonic survival. 45
Figure 12. Maternal zygotic nrz mutants exhibit epiboly arrest phenotype. 46
Figure 13. Progression of blastoderm margin constriction in gastrulating MZnrzd7/d7 embryos. 47
Figure 14. Maternal and zygotic deficiency of Nrz on embryonic development. 48
Figure 15. Effects genetic background of mutant nrz on the initiation of blastoderm margin constriction. 50
Figure 16. Rescue of epiboly defects in MZnrz mutant embryos by injecting nrz mRNA. 54
Figure 17. Intensity profiles of phalloidin signal in MZnrzi3d1/i3d1 embryos. 55
Appendix Figures 56
Appendix Figure 1. The expression level of nrz at shield stage in wild type and MZnrz mutant embryos. 56
Appendix Figure 2. The actin ring formation of nrz mutant embryos. 57
Video Captions 58
Video 1. Epiboly arrest of MZnrzd7/d7 embryo at shield stage. 58
Video 2. Epiboly arrest of MZnrzi3d1/i3d1 embryo at dome stage. 58
Video 3. Premature constriction of MZnrzd7/d7 embryo at high stage. 58
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