向小鼠导入α-1，3-GT沉默基因及其整合效果的研究
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摘要
为了沉默α-1,3-半乳糖苷转移酶,在已报道的shRNA序列前加上人类H1启动子,设计一段沉默小鼠α-1,3-GT基因的siRNA序列。针对该序列分段设计引物,完全采用PCR法将其连接起来,再把该序列克隆到pGEM-T载体中,构建出针对小鼠α-1,3-GT基因的特异性沉默载体。将其酶切线性化后通过显微注射法导入小鼠受精卵中,然后进行胚胎移植。最后利用PCR和Southern blot对出生仔鼠进行了外源基因的整合检测。该研究为获得α-1,3-GT基因沉默小鼠,构建异种器官移植用小鼠模型奠定了重要基础。
1、经PCR法和测序验证,已经顺利用两步PCR法将设计的引物连接起来,并成功的克隆进入pGEM-T载体中。
2、利用显微注射法将线性化的α-1,3-GT基因沉默载体导入2846枚小鼠受精卵中,得到可移胚2291枚,将其移入76只受体母鼠,其中有35只母鼠妊娠,妊娠率为46.1%。在母鼠妊娠过程中,前后有29只母鼠流产,未流产的6只妊娠小鼠产仔21只。
3、在显微注射时,观察了胚胎移植数及不同细胞期对产仔率的影响。结果表明:受体小鼠移植21枚~30枚显微注射胚可以获得较高的产仔率;移植二细胞期的受体小鼠妊娠率及产仔率明显高于移植一细胞期卵的受体小鼠。
4、利用PCR和Southern blot对21只活鼠和17个死鼠(包括死胎、木乃伊)进行了外源基因的整合检测,一共检测到7个阳性个体(三只活鼠,其它为死胎、木乃伊),外源基因整合率为14.3%。
Through putting human H1 promoter before the shRNA reported,a siRNA sequence was designed to silenceα-1,3-GT gene in murine. Subsequently a series of primer segments were devised and joined by PCR.After the connected product was cloned into the pGEM-T vector, a vector that can silence the murineα-1,3-GT gene was acquired.After linearization by enzym e digestion,the vector silencingα-1,3-GT gene was transferred into murine fertilized eggs and carry out embryo transplantation.Finally,the integration of exogenous gene was examined by PCR and Southern blot analysis after the birth of young mice.This reasearch will help us to constitute an important foundation for acquiringα-1,3-GT knock down mice and mouse model for xenotransplantation research.
1.Fragments of devised primer have been joined successfully by two-step PCR and cloned into pGEM-T vector by PCR and sequencing.
2.Linear vector for silencingα-1,3-GT gene was microjected into 2846 of murine ferti- lized eggs and 2291 of useful embryos were transferred into 76 of recipients.35 of them had pregnancies.The pregnant is 46.1%.There were 29 of mother mice aborting and the others giving birth to 21 offsprings in the gestation period.
3.The influence that the number of embryo and different cell stages acted on lambing rate was observed in the process of microjection.The results showed:the group of 21-30 embryos have significantly higher dropping rate than other groups;Two-cell stage has signifi- cantly higher conception rate and dropping rate than one-cell stage.
4.The integration of exogenous gene was examined in the 21 of live mice and 17 of dead mice(including fetal death and mummy)by PCR and Southern blot analysis. Seven of positive mice were observed including three of live mice and four of dead mice.The total integrated rate of exogenous gene is 14.3%
1、经PCR法和测序验证,已经顺利用两步PCR法将设计的引物连接起来,并成功的克隆进入pGEM-T载体中。
2、利用显微注射法将线性化的α-1,3-GT基因沉默载体导入2846枚小鼠受精卵中,得到可移胚2291枚,将其移入76只受体母鼠,其中有35只母鼠妊娠,妊娠率为46.1%。在母鼠妊娠过程中,前后有29只母鼠流产,未流产的6只妊娠小鼠产仔21只。
3、在显微注射时,观察了胚胎移植数及不同细胞期对产仔率的影响。结果表明:受体小鼠移植21枚~30枚显微注射胚可以获得较高的产仔率;移植二细胞期的受体小鼠妊娠率及产仔率明显高于移植一细胞期卵的受体小鼠。
4、利用PCR和Southern blot对21只活鼠和17个死鼠(包括死胎、木乃伊)进行了外源基因的整合检测,一共检测到7个阳性个体(三只活鼠,其它为死胎、木乃伊),外源基因整合率为14.3%。
Through putting human H1 promoter before the shRNA reported,a siRNA sequence was designed to silenceα-1,3-GT gene in murine. Subsequently a series of primer segments were devised and joined by PCR.After the connected product was cloned into the pGEM-T vector, a vector that can silence the murineα-1,3-GT gene was acquired.After linearization by enzym e digestion,the vector silencingα-1,3-GT gene was transferred into murine fertilized eggs and carry out embryo transplantation.Finally,the integration of exogenous gene was examined by PCR and Southern blot analysis after the birth of young mice.This reasearch will help us to constitute an important foundation for acquiringα-1,3-GT knock down mice and mouse model for xenotransplantation research.
1.Fragments of devised primer have been joined successfully by two-step PCR and cloned into pGEM-T vector by PCR and sequencing.
2.Linear vector for silencingα-1,3-GT gene was microjected into 2846 of murine ferti- lized eggs and 2291 of useful embryos were transferred into 76 of recipients.35 of them had pregnancies.The pregnant is 46.1%.There were 29 of mother mice aborting and the others giving birth to 21 offsprings in the gestation period.
3.The influence that the number of embryo and different cell stages acted on lambing rate was observed in the process of microjection.The results showed:the group of 21-30 embryos have significantly higher dropping rate than other groups;Two-cell stage has signifi- cantly higher conception rate and dropping rate than one-cell stage.
4.The integration of exogenous gene was examined in the 21 of live mice and 17 of dead mice(including fetal death and mummy)by PCR and Southern blot analysis. Seven of positive mice were observed including three of live mice and four of dead mice.The total integrated rate of exogenous gene is 14.3%
引文
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