The Study On Integration And Expression Of HMCP And HCD59 Genes Microinjected To Mice

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.