本文選題：CPED1 + 雞��； 參考：《揚州大學》2017年碩士論文

【摘要】：精原干細胞(spermatogonial stem cells,SSCs)是動物體內重要的成體干細胞,能夠將遺傳物質傳遞給下一代,它既有胚胎干細胞的發(fā)育特性又能發(fā)育成單倍體生殖細胞�，F(xiàn)今有大量研究表明精原干細胞可在體外被誘導為具有不同功能的細胞系,用于遺傳修飾、疾病治療和轉基因動物制備等,因此對精原干細胞體外操作的研究尤為重要。其中將SSCs誘導形成精子一直是科學家們研究的熱門話題,但如何大量獲得SSCs并使其產生具有功能性的精子就成為科學家們亟需解決的科學問題。通過本課題組前期已完成的雞雄性ESCs,雄性PGCs以及SSCs的轉錄組測序(RNA-Seq)的實驗結果,分析得到了雞雄性生殖細胞發(fā)生過程中基因動態(tài)表達的水平變化,有助于對該過程中重要作用的未知基因的發(fā)現(xiàn)。本實驗研究的目的基因CPED1在SSCs的轉錄組測序中特異高表達并功能未知,因此有可能對SSCs分化具有重要作用,因此本實驗同時利用基因過表達技術和CRISPR/Cas9技術實現(xiàn)對測序過程中發(fā)現(xiàn)的精原干細胞高表達基因CPED1功能的研究,為闡明生殖細胞發(fā)生及分化機制提供高效方法。本研究的主要內容如下：(1)基于本實驗室前期RNA-Seq技術發(fā)現(xiàn)CPED1在ESCs向SSCs分化過程中存在顯著差異表達。通過qPCR克隆如皋黃雞CPED1編碼序列,同時構建過表達載體pcDNA3.0-CPED1和敲除載體Cas9/gRNA。通過Fugene轉染Cas9/gRNA載體,利用T7E1酶切、SSA活性檢測、TA克隆測序和脫靶效率檢測Cas9/gRNA載體敲除活性及脫靶情況。在DF-1細胞中通過T7E1酶切檢測Cas9/gRNA載體的活性,酶切結果表明,通過條帶灰度值估計Cas9/gRNA1載體、Cas9/gRNA2載體和Cas9/gR/A3載體活性分別為377、20%和30%%,Ca9/gR/A1載體敲除活性最佳;SSA活性檢測結果表明gRNA1的熒光活性值與對照組相比增加了 2倍左右,活性最佳;TA克隆測序結果表明測序的8菌液樣中有2管菌液樣發(fā)生了突變,初步估計基因敲除率為25%左右;脫靶效率檢測結果表明在DF-1細胞中無脫靶現(xiàn)象。同時實現(xiàn)了在ESCs中CPED1的定點敲除,敲除效率為25%。這一結果表明CRISPR/Cas9技術能穩(wěn)定的在雞DF-1細胞和ESCs上實現(xiàn)CPED1敲除。(2)分別將Cas9/gRNA1載體和pcDNA3.0-CPED1過表達載體通過Fugene轉染ESCs,轉染48小時候換RA誘導培養(yǎng)基培養(yǎng)12d。采用細胞形態(tài)學觀察、間接免疫熒光檢測、流式細胞分析和qRT-PCR等方法檢測CPED1于敲除與過表達對ESCs向SSCs分化的影響,結果表明敲除CPED1的ESCs與正常RA誘導組相比,類胚體形成時間延長且停止向雄性生殖細胞分化;第12dRA誘導組與過表達組均能形成類SSCs,敲除組則無類SSCs形成。間接免疫熒光檢測結果表明敲除CPED1導致體外誘導10d的ESCs分化生成的integrin α6和integrin β1雙陽性類SSCs數(shù)量顯著低于過表達組和RA誘導組。流式細胞分析結果表明RA誘導組、過表達組和敲除組中integrin α6陽性類SSCs數(shù)量比例分別為1.5%±0.163、1.8%%±0.294和0.9%±0.216,敲除組中陽性細胞率明顯低于其它兩組。12d qRT-PCR結果表明RA誘導組中目的基因CPED1和生殖相關基因Cvh、C-kit、Stra8、integrin α6和integrin β1的最高表達量分別為 1.72,1.62,1.74,2.01,2.36,2.42;過表達組中目的基因CPED1和生殖相關基因Cvh、C-kit、Strt8、integrinα6和integrin β1的最高表達量分別為2.68,1.71,1.95,2.46,2.67,2.78;敲除組中目的基因CPED1和生殖相關基因Cvh、C-kit、Stra8、integrin α6和integrin β1的最高表達量分別為 0.42,1.12,1.03,0.87,1.41,1.53;RA誘導組和過表達組中CPED1的表達量具有顯著差異,敲除組中CPED1以及生殖相關基因Cvh、C-kit、、Stra8、integrin α6和integrin β1的表達量與RA誘導組和過表達組相比呈顯著下調。在RA誘導ESCs向SSCs分化過程中,敲除CPED1抑制類SSCs形成,證明CPED1在調控家禽ESCs向SSCs分化過程中起重要作用。(3)取新鮮的受精雞胚,利用PEI包裹Cas9/gRNA1載體和pcDNA3.0-CPED1過表達載體分別進行雞胚注射,并設立正常孵化組與對照組。分別收集正常孵化至4.5d雞胚生殖嵴樣和18.5d睪丸樣,采用石蠟切片及PAS染色、qRT-PCR和流式細胞分析等方法檢測CPED1敲除和過表達對雞雄性生殖細胞體內動態(tài)變化的影響。qRT-PCR檢測結果顯示4.5d的過表達組中生殖相關基因Cvh、Stra8和integrin α6的表達量分別為2.12,1.37,1.20,顯著高于敲除組,CPED1表達量(1.07)極顯著高于敲除組;18d的過表達組中的Nanog的表達量(0.32)顯著低于對照組,integrin α6基因表達量(1.89)顯著高于敲除組,CPED1表達量(2.09)極顯著高于敲除組。4.5d雞胚石蠟切片結果表明對照組中PGCs數(shù)量為(45±2.236)個,過表達組中PGCs數(shù)量為(41±1.699)個,敲除組中PGCs數(shù)量為(18±0.745)個,敲除組中含有的PGCs數(shù)量低于對照組和過表達組。同時對18d睪丸分離培養(yǎng)的SSCs進行流式細胞分析檢測,結果表明對照組、過表達組和敲除組中integrin α6陽性細胞率分別為1.3%±0.141、1.6%±0.356和0.8%±0.245,敲除組中SSCs數(shù)量顯著減少,對照組和過表達組中卻無顯著差異。結果證明CPED1對雞胚干細胞向精原干細胞分化具有促進作用。
[Abstract]:Spermatogonial stem cells (SSCs) is an important adult stem cell in the animal. It can transmit genetic material to the next generation. It has both the developmental characteristics of embryonic stem cells and the development of haploid germ cells. There is a lot of research now that spermatogonial stem cells can be induced in vitro into different functional cell lines. The research on the operation of spermatogonial stem cells in vitro is particularly important for genetic modification, disease treatment and the preparation of genetically modified animals. It has been a hot topic for scientists to induce the formation of spermatozoa by SSCs, but how to obtain SSCs and make it produce functional sperm has become a science that scientists need to solve. Problems. Through the experimental results of the male ESCs, male PGCs and SSCs transcriptional sequence (RNA-Seq), the changes of gene dynamic expression during the development of the male reproductive cells of the chicken were analyzed, which was helpful to the discovery of the unknown genes that played an important role in the process. The target gene of this experiment was CPE. D1 is highly expressed in SSCs transcriptional sequence and has unknown function. Therefore, it may play an important role in the differentiation of SSCs. Therefore, this experiment also uses gene overexpression technology and CRISPR/Cas9 technology to study the function of high expression gene CPED1 of spermatogonial stem cells found in the sequencing process, in order to clarify the pathogenesis and differentiation machine of germ cells. The main contents of this study are as follows: (1) there is a significant difference in the expression of CPED1 during the differentiation of ESCs to SSCs in the pre laboratory RNA-Seq technology. The CPED1 coding sequence of Rugao yellow chicken was cloned by qPCR, and the expression vector pcDNA3.0-CPED1 and the knockout carrier Cas9/gRNA. were constructed by Fugene transfection Cas9/gRNA. Vector, using T7E1 enzyme cutting, SSA activity detection, TA cloning sequencing and miss efficiency detection of Cas9/gRNA vector knockout activity and miss target. The activity of Cas9/gRNA vector was detected by T7E1 enzyme digestion in DF-1 cells. The results showed that Cas9/gRNA1 vector was estimated by the gray value of strip and the activity of Cas9/gRNA2 carrier and Cas9/gR/A3 carrier was 377,2. 0% and 30%%, Ca9/gR/A1 carrier knockout activity was the best, SSA activity detection results showed that the fluorescence activity of gRNA1 increased about 2 times compared with the control group, and the activity was the best. The result of TA cloning sequencing results showed that there was a mutation of 2 tube samples in the sequence of 8 bacteria samples, and the initial estimated gene knockout rate was about 25%. There is no dislocation in DF-1 cells. At the same time, the targeted knockout of CPED1 in ESCs and the knockout efficiency of 25%. show that CRISPR/Cas9 technology can stabilize the CPED1 knockout on chicken DF-1 cells and ESCs. (2) the Cas9/gRNA1 carrier and pcDNA3.0-CPED1 overexpression vector pass through Fugene transfection ESCs, and the transfection 48 When transfection is induced to induce culture. Cell morphology observation, indirect immunofluorescence detection, flow cytometry and qRT-PCR were used to detect the effect of CPED1 on the differentiation of ESCs to SSCs by CPED1. The results showed that the ESCs of knockout CPED1 was longer than normal RA induction group and stopped to the male reproductive cells. 12dRA Both the induced group and the overexpressed group could form a class of SSCs, and the knockout group had no SSCs like formation. The results of indirect immunofluorescence showed that the number of integrin alpha 6 and integrin beta 1 positive class SSCs generated by the knockout of CPED1 in vitro induced the differentiation of 10d was significantly lower than that of the overexpressed and RA induced groups. The proportion of integrin alpha 6 positive SSCs in the group and the knockout group were 1.5% + 0.163,1.8%% + 0.294 and 0.9% + 0.216 respectively. The positive cell rate in the knockout group was significantly lower than the other two groups of.12d qRT-PCR results showed that the target gene CPED1 and the reproductive related gene Cvh, C-kit, Stra8, integrin alpha 6 and integrin beta 1 in the RA induction group were the highest levels of expression. The highest expression of the target gene CPED1 and reproductive related genes Cvh, C-kit, Strt8, integrin alpha 6 and integrin beta 1 in the overexpressed group was 2.68,1.71,1.95,2.46,2.67,2.78, and the highest expression of the target gene CPED1 and reproductive related genes Cvh, C-kit, Stra8, alpha 6 and C-kit beta 1 in the knockout group The amount of CPED1 expression in RA induced and overexpressed groups was significantly different. The expression of CPED1 and reproductive related genes Cvh, C-kit, Stra8, integrin alpha 6 and integrin beta 1 in the knockout group were significantly down regulated by RA induced and overexpressed groups. In addition to the formation of CPED1 inhibitory SSCs, CPED1 plays an important role in regulating the differentiation of poultry ESCs into SSCs. (3) take fresh fertilized chicken embryos, use PEI to encapsulate Cas9/gRNA1 vector and pcDNA3.0-CPED1 overexpression vector for chicken embryo injection respectively, and set up normal hatching group and control group, and collect normal hatching to 4.5d chicken embryo reproductive crest samples, respectively. The effects of CPED1 knockout and over expression on the dynamic changes of the male reproductive cells in the chickens were detected by paraffin section and PAS staining, qRT-PCR and flow cytometry. The results of.QRT-PCR detection showed that the expression of reproductive related genes Cvh, Stra8 and integrin 6 were 2.12,1.37,1.20, respectively, in the overexpressed group of 4.5d. The results showed that the expression of Cvh, Stra8 and integrin 6 were 2.12,1.37,1.20, respectively. The expression of CPED1 (1.07) was significantly higher than that in knockout group, and the expression of Nanog in the overexpression group of 18D was significantly lower than that of the control group. The expression of integrin alpha 6 gene (1.89) was significantly higher than that in the knockout group, and the CPED1 expression (2.09) was significantly higher than that of the knockout group.4.5d chicken embryo paraffin section, indicating that the number of PGCs in the control group was (45 + 2.236). The number of PGCs in the overexpression group was (41 + 1.699), and the number of PGCs in the knockout group was (18 + 0.745). The number of PGCs contained in the knockout group was lower than that of the control group and the overexpressed group. At the same time, the SSCs of the 18D testis isolated and cultured was detected by flow cytometry. The results showed that the ratio of integrin alpha 6 positive cells in the over expression group and the knockout group was in the control group. It was 1.3% + 0.141,1.6% + 0.356 and 0.8% + 0.245. The number of SSCs decreased significantly in the knockout group, but there was no significant difference between the control group and the overexpressed group. The results showed that CPED1 could promote the differentiation of chick embryonic stem cells to spermatogonial stem cells.

【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S831

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