向小鼠导入人类MCP和CD59基因的整合和表达研究
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摘要
本研究在探索了提高小鼠超数排卵效果的基础上，利用显微原核注射技术将hMCP和hCD59基因导入小鼠受精卵中，采用PCR和Southern blot对仔鼠进行了外源基因的整合检测，并利用细胞毒实验对检测到的转基因阳性小鼠进行了表达分析。结果如下：
1. 分析了激素剂量、激素注射间隔时间、发情周期、季节和温度对小鼠超排效果的影响。当PMSG与hCG剂量为5-10IU时，见栓小鼠平均获卵数差异不显著（P>0.05）， 激素剂量为15IU时，见栓小鼠平均获卵数显著下降(P<0.05)；激素注射间隔时间46h与48h见栓小鼠平均获卵数差异不显著(P>.05)，当激素注射时间间隔为50h时，见栓小鼠平均获卵数显著下降(P<0.05)；在发情周期不同阶段超排效果不同，发情前期开始超排，其见栓率、见栓小鼠平均获卵数均显著高于其他情期(P<0.05)；当环境温度维持在20±5℃时，不同季节小鼠超排效果无显著差异(P>0.05)；在冬季，鼠房温度为20±2℃时，小鼠超排见栓率和见栓小鼠平均获卵数均极显著高于环境温度为10±2℃的超排效果(P<0.01)。
2. 利用显微注射技术将hMCP和hCD59基因导入2153枚小鼠受精卵中，其中可移胚为1725枚，移植到62只受体母鼠，其中有29只母鼠妊娠，妊娠率为46.7%。在母鼠妊娠过程中，前后有13只母鼠流产，流产母鼠解剖后，观察发现子宫内有胚胎附着点存在。未流产的16只妊娠小鼠产仔71只，可移胚的产仔率为4.1%（71/1725）。
3.本研究分析了导入有hMCP和hCD59基因小鼠胚胎的移植数量和分别在左侧或右侧输卵管胚胎移植对受体母鼠产仔率和妊娠率的影响，结果表明，移入21-30枚注射胚数量组的产仔率和妊娠率均极显著高于移入10-20枚注射胚数量的产仔率和妊娠率 (P<0.01)；移入21-30枚注射胚数量组的妊娠率与移入31-45枚数量组的妊娠率相比，差异不显著（P>0.05）；移入21-30枚注射胚数量组的产仔率显著高于移入31-45枚组的产仔率（P<0.05）；在受体小鼠左侧或右侧输卵管移植胚胎的妊娠率和产仔率差异不显著（P>0.05）。
4.利用PCR和Southern blot对71只仔鼠进行了外源基因的整合检测，一共检测到6只阳性小鼠，其中4只为hCD59基因阳性小鼠（活仔），2只为hMCP基因阳性小鼠（死胎）。外源基因整合率为8.5%（6/71）。
5.利用细胞毒实验对4只hCD59基因阳性小鼠进行了表达分析，发现有3只小鼠淋巴细胞对混合人血清有不同程度的抗性，证明这3只小鼠表达有人的CD59分子，并且hCD59基因的表达产物能在一定程度上抑制超急性排斥反应。
On the base of improvement of mouse superovulation, hCD59 gene and hMCP gene were microinjected to mouse zygote in this study. The integration of hCD59 gene and hMCP gene in offspring mice were examined through PCR and Southern blot analysis and the expression of foreign gene in observed positive mouse was studied through microlymphocytotoxicity. Results were as follows:
1. Effects of hormone dosage, interval time of hormone injection, oestrus period, season and temperature influenced on mouse superovulation were studied in this experiment. When the dosage of PMSG and HCG were 5-10IU, average embryos of mated mouse have no significant difference(P>0.05) but significant higher than the dosage of 15IU(P<0.05).The average embryos of mated mouse between 46h and 48h of hormone injection interval time had no distinct difference(p>0.05). The average embryo of mated mouse was decreased distinctly when the interval time of hormone injection was 50h (p<0.05). The mouse treated in different oestrus period had different superovulation results. The rate and embryos of mated mouse were significant higher than other oestrus period (P<0.01). There was no significant difference among different seasons if the environment temperature maintained 20±5℃(P>0.05).In winter, rate and average embryos of mated mouse treated under condition of 20±2℃ environment temperature were significant higher than the mouse treated under condition of 10±2℃ environment temperature(p<0.01).
2.In this study, 2153 mouse zygotes were transferred hCD59 gene and hMCP gene through microinjection. 1725 zygotes survived after microinjection and were transplanted to 62 recipient female mice.29 recipients were pregnancy and the pregnancy rate was 46.7%.13 recipients were aborted during pregnancy period. Attachment spots were observed in uterus when aborted mice were dissected. The rest of pregnancy mice gave birth to 71 offspring and the lambing rate was 4.1%.
3. The effects of the Number of zygotes microinjected hCD59 gene and hMCP gene and zygotes transplanted to left side oviduct or right side oviduct on the lambing rate and pregnancy rate were analyzed in this study. Results showed: The group of 21-30 embryos was observed significant higher lambing rate and pregnancy rate than 10-20 embryos group (p<0.01). The pregnancy rate between 31-45 embryos and 21-30 embryos had no distinct difference(p>0.05); The lambing rate of 21-30 embryos group was distinct higher than 31-45 embryos group(p<0.05).There were no significant difference on pregnancy rate and lambing rate between left-side embryos transplanted group and right side embryos transplanted
group(p>.05).
4. The integration of CD59 gene and MCP gene were examined in 71 offspring mice through PCR and Southern blot analysis. Six positive mice were observed. Four positive mice have hCD59 gene integrated. Two positive mice have hMCP gene integrated. The total integrated rate of foreign gene is 8.5%.
5. The expression of CD59 gene in four positive mice was analyzed through microlymphocytotoxicity. Three mice’s lymphocytes had the ability to resist to human serum, which proved that these mice had expressed human CD59.
1. 分析了激素剂量、激素注射间隔时间、发情周期、季节和温度对小鼠超排效果的影响。当PMSG与hCG剂量为5-10IU时，见栓小鼠平均获卵数差异不显著（P>0.05）， 激素剂量为15IU时，见栓小鼠平均获卵数显著下降(P<0.05)；激素注射间隔时间46h与48h见栓小鼠平均获卵数差异不显著(P>.05)，当激素注射时间间隔为50h时，见栓小鼠平均获卵数显著下降(P<0.05)；在发情周期不同阶段超排效果不同，发情前期开始超排，其见栓率、见栓小鼠平均获卵数均显著高于其他情期(P<0.05)；当环境温度维持在20±5℃时，不同季节小鼠超排效果无显著差异(P>0.05)；在冬季，鼠房温度为20±2℃时，小鼠超排见栓率和见栓小鼠平均获卵数均极显著高于环境温度为10±2℃的超排效果(P<0.01)。
2. 利用显微注射技术将hMCP和hCD59基因导入2153枚小鼠受精卵中，其中可移胚为1725枚，移植到62只受体母鼠，其中有29只母鼠妊娠，妊娠率为46.7%。在母鼠妊娠过程中，前后有13只母鼠流产，流产母鼠解剖后，观察发现子宫内有胚胎附着点存在。未流产的16只妊娠小鼠产仔71只，可移胚的产仔率为4.1%（71/1725）。
3.本研究分析了导入有hMCP和hCD59基因小鼠胚胎的移植数量和分别在左侧或右侧输卵管胚胎移植对受体母鼠产仔率和妊娠率的影响，结果表明，移入21-30枚注射胚数量组的产仔率和妊娠率均极显著高于移入10-20枚注射胚数量的产仔率和妊娠率 (P<0.01)；移入21-30枚注射胚数量组的妊娠率与移入31-45枚数量组的妊娠率相比，差异不显著（P>0.05）；移入21-30枚注射胚数量组的产仔率显著高于移入31-45枚组的产仔率（P<0.05）；在受体小鼠左侧或右侧输卵管移植胚胎的妊娠率和产仔率差异不显著（P>0.05）。
4.利用PCR和Southern blot对71只仔鼠进行了外源基因的整合检测，一共检测到6只阳性小鼠，其中4只为hCD59基因阳性小鼠（活仔），2只为hMCP基因阳性小鼠（死胎）。外源基因整合率为8.5%（6/71）。
5.利用细胞毒实验对4只hCD59基因阳性小鼠进行了表达分析，发现有3只小鼠淋巴细胞对混合人血清有不同程度的抗性，证明这3只小鼠表达有人的CD59分子，并且hCD59基因的表达产物能在一定程度上抑制超急性排斥反应。
On the base of improvement of mouse superovulation, hCD59 gene and hMCP gene were microinjected to mouse zygote in this study. The integration of hCD59 gene and hMCP gene in offspring mice were examined through PCR and Southern blot analysis and the expression of foreign gene in observed positive mouse was studied through microlymphocytotoxicity. Results were as follows:
1. Effects of hormone dosage, interval time of hormone injection, oestrus period, season and temperature influenced on mouse superovulation were studied in this experiment. When the dosage of PMSG and HCG were 5-10IU, average embryos of mated mouse have no significant difference(P>0.05) but significant higher than the dosage of 15IU(P<0.05).The average embryos of mated mouse between 46h and 48h of hormone injection interval time had no distinct difference(p>0.05). The average embryo of mated mouse was decreased distinctly when the interval time of hormone injection was 50h (p<0.05). The mouse treated in different oestrus period had different superovulation results. The rate and embryos of mated mouse were significant higher than other oestrus period (P<0.01). There was no significant difference among different seasons if the environment temperature maintained 20±5℃(P>0.05).In winter, rate and average embryos of mated mouse treated under condition of 20±2℃ environment temperature were significant higher than the mouse treated under condition of 10±2℃ environment temperature(p<0.01).
2.In this study, 2153 mouse zygotes were transferred hCD59 gene and hMCP gene through microinjection. 1725 zygotes survived after microinjection and were transplanted to 62 recipient female mice.29 recipients were pregnancy and the pregnancy rate was 46.7%.13 recipients were aborted during pregnancy period. Attachment spots were observed in uterus when aborted mice were dissected. The rest of pregnancy mice gave birth to 71 offspring and the lambing rate was 4.1%.
3. The effects of the Number of zygotes microinjected hCD59 gene and hMCP gene and zygotes transplanted to left side oviduct or right side oviduct on the lambing rate and pregnancy rate were analyzed in this study. Results showed: The group of 21-30 embryos was observed significant higher lambing rate and pregnancy rate than 10-20 embryos group (p<0.01). The pregnancy rate between 31-45 embryos and 21-30 embryos had no distinct difference(p>0.05); The lambing rate of 21-30 embryos group was distinct higher than 31-45 embryos group(p<0.05).There were no significant difference on pregnancy rate and lambing rate between left-side embryos transplanted group and right side embryos transplanted
group(p>.05).
4. The integration of CD59 gene and MCP gene were examined in 71 offspring mice through PCR and Southern blot analysis. Six positive mice were observed. Four positive mice have hCD59 gene integrated. Two positive mice have hMCP gene integrated. The total integrated rate of foreign gene is 8.5%.
5. The expression of CD59 gene in four positive mice was analyzed through microlymphocytotoxicity. Three mice’s lymphocytes had the ability to resist to human serum, which proved that these mice had expressed human CD59.
引文
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