臺灣博碩士論文加值系統

性別決定的機制在細胞生長發育中扮演重要角色，其中PHD finger protein 7(Phf7)基因是調控雄性生殖細胞發育及生長的重要基因。在果蠅中，Phf7在早期雄性生殖細胞，雄性生殖幹細胞和精原細胞中具有專一性的表現，Phf7 基因會影響雄性生殖細胞產生精子的能力。
在我們的實驗中，目的是尋找與Phf7共同參與在果蠅生殖細胞生長發育的未知基因。首先，我們使用Minos transposon隨機鑲嵌到果蠅基因中破壞基因功能，觀察過量表現Phf7在雌性生殖細胞的果蠅是否能恢復產生後代的能力。結果顯示，部分雌果蠅在沒有Minos transposon 作用產生突變下，仍然可以從Phf7對於雌性生殖細胞的破壞中恢復，並且具有產生後代的能力。
在另一實驗中，我們發現在第三對染色體部分缺失加上Phf7 剔除(Phf7△88 )之雄果蠅生殖腺具有不同的表現型。目前實驗發現，在CG7907 基因突變加上Phf7 剔除 (Phf7△18)之雄果蠅生殖腺有部分不同於wild type的表現型；而在免疫螢光染色結果中也顯示早期生殖細胞與hub cell距離有靠近的趨勢，顯示將這兩個基因同時剔除可能影響早期生殖細胞生長發育。因此，我們認為CG7907 基因可能是與Phf7基因共同參與在果蠅雄性生殖細胞發育的候選基因。

Sex determination in soma and germline are different. Both of them need critical regulation to produce gametes. An important gene which regulates male germline development is PHD finger protein 7 (Phf7). In Drosophila, Phf7 is specifically expressed in the early male germline, male germline stem cells, and spermatogonia. Phf7 expression is important for germline stem cell maintenance and gametogenesis in males.
Here, we design an experiment to find the unknown genes that participate in sex determination together with Phf7 in the drosophila germline. We use a Minos tranposon to randomly integrate in the Drosophila genome to disrupt gene function. Subsequently, we can screen for flies that can produce progeny which likely results from transposon integration into a gene that can rescue the germline loss caused by Phf7 overexpression. Our current results show that the frequency of transposon remobilization is too low. Strangely, some Drosophila produced partially-rescued ovaries without transposon-mediated mutagenesis.
In another screening, based on the previous results, we found oval testis phenotypes in (〖"Phf7" 〗^"△N18" )/"y" ；"Df" ("3R" )"ED6058" /"+" Drosophila. According to the current results, we found that there is an abnormal winding testis phenotype in (〖"Phf7" 〗^"△N18" )/"y" ；〖"CG7907" 〗^"MI15586" /"+" Drosophila. Immunofluorescence staining results showed that the early germ cells and hub cell distance get close in (〖"Phf7" 〗^"△N18" )/"y" ；〖"CG7907" 〗^"MI15586" /"+" Drosophila. Therefore, we think that CG7907 gene may be a candidate gene that act with Phf7 gene and involve in the Drosophila male germline developement.

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Acknowledgement iii
中文摘要 iv
Abstract v
Content vi
List of Figures viii
List of Tables ix
Chapter 1 Introduction - 1 -
1.1. Sex determination - 1 -
1.2. Sex determination in Drosophila somatic cells - 2 -
1.3. Sex determination in Drosophila germ cells - 3 -
1.4. Drosophila Phf7 in sex development - 5 -
1.5. Drosophila PHF7 is a histone reader protein - 6 -
1.6. Phf7 requires other factor in male germ cells sexual identity - 6 -
Chapter 2 Experiment design - 8 -
Chapter 3 Experimental Materials and methods - 9 -
3.1 Fly strains - 9 -
3.2 Single fly DNA extraction - 10 -
3.3 DNA extraction - 10 -
3.4 Single fly PCR - 11 -
3.5 Drosophila ovary dissection - 11 -
3.6 PCR - 12 -
3.7 Immunofluorescence staining for adult testes - 12 -
Chapter 4 Results - 14 -
4.1. Screening of new candidate genes in Drosophila germline sex determination - 14 -
4.2. Optimization of transposition efficiency - 16 -
4.3. Minos transposon interfere Phf7 overexpression in female germline - 17 -
4.4. Efficiency of MB01657 transposon is still low - 18 -
4.5. Chromosome deficiency kit screening to identify - 18 -
Phf7-related genes - 18 -
4.6. CG7907 may be a candidate gene that act with Phf7 gene in Drosophila male germline development - 20 -
4.7. Immunofluorescence staining of germ cells to investigate germline division - 23 -
Chapter 5 Discussion - 25 -
Chapter 6 Figure - 32 -
Chapter 7 Reference - 43 -

List of Figures
Figure 1. 5 day-old ovaries overexpressing Phf7 in the germline with additional Minos
transposon insertions....................................................................................................- 32 -
Figure 2. Minos transposition efficiency in two heat shock programs ........................- 33 -
Figure 3. 3 day-old to 9 day-old ovaries overexpressing Phf7 in the germline without
additional Minos transposon insertions........................................................................- 34 -
Figure 4. Minos transposition efficiency in 2 hours heat shock programs...................- 35 -
Figure 5. Chromosome defect region in 3R chromosome deficiency kit Drosophila and
testes phenotype of deficiency Drosophila cross with Phf7 deletion Drosophila.. ......- 36 -
Figure 6. Phenotype of offspring testis between wild-type and 3R chromosome
deficiency male Drosophila cross with Phf7
△N18 female Drosophila.. .........................- 37 -
Figure 7. CG7907 gene expression between Oregon R and CG7907 RNAi
(CG7907HMC04058)..........................................................................................................- 38 -
Figure 8. Effects of CG7907 mutation, Phf7 deletion (Oregon R x Phf7Δ18), CG7907
mutation plus Phf7 deletion, Df(3R)ED6058 male Drosophila cross with Phf7△N18 female
Drosophila on Germ Cells............................................................................................- 39 -

List of Tables
Table 1. Amounts of drosophila in Minos transposon interfere the effect of Phf7
overexpression in female germline ...................................................................- 40 -
Table 2. Amounts of mutant testes in CG7907 gene mutant, Phf7 deletion and Phf7
deletion plus deficiency kit.................................................................................- 41 -
Table 3. Primers data of PCR experiment..........................................................- 42 -

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