| Cytoplasmic microinjection-mediated gene transfer (CI-MGT) has the advantages of simple to operate, little embryo damage and short persistence time in vitro. Recently, the combination of CI and in vivo fertilizated embryos with efficient genome editing technology has been widely used for transgenic animal production. Intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) is especially suitable for transgenic pig production, because of it is useful for large DNA fragment transfer, and it can efficiently resolve the problem of polyspermic penetration during in vitro fertilization (IVF). Guangxi is located in the high temperature and humidity subtropical region, and to optimize pig embryo production and transgenic technology of this environment, the current study on the basis of optimization of porcine oocytes in vitro mature (IVM), parthenogenetic activation (PA) and in vitro culture (IVC) conditions, factors influencing of PA and IVF embyros CI-MGT and ICSI-MGT were systematically investigated, and the feasibility of using IVF CI-MGT and ICSI-MGT embryos to product transgenic pig was also evaluated. Main results for this study are as follows:1. Components of culture medium on IVM and IVC were systematically investigated. The results indicated, the nuclear maturation rate and embyro development rate of IVM basic medium TCM-199 were significantly higher than that of NCSU-37 and PZM-3. TCM-199 as the IVM basic medium, nuclear maturation rate and embyro development rate of no addition extra energy substrates (PMt) were 74.1±2.4% and 29.3±1.3% respectively, and it was significantly higher than that of added 0.91 mmol/L sodium pyruvate+3.05 mmol/L glucose (PMts) or 0.91 mmol/L sodium pyruvate+3.05 mmol/L glucose+O.lmg/mL glutamine (PMtsgg, P<0.05). The nuclear maturation rate and embyro development rate of added 10 IU/mL eCG+15 IU/mL hCG was significantly higher than that of 0.5 μg/mL FSH+0.5μg/mL LH,0.5 μg/mL FSH+10 UI/mL hCG Kinetics of in vitro nuclear maturation indicated, the proportions of oocytes reached MII stage at 38 h 57%(105/184), at 40 h 43%(82/189), at 42 h 48%(75/155), at 44 h 59%(208/354), at 46 h 70%(59/84), at 48 h 62%(51/82), at 50 h 74%(154/207).IVC medium optimization was based on the PZM-3, the blastocyst rate of added 0.277 mmol/L inositol (PZMi) was significantly higher than that of added 0.34 mmol/L sodium citrate (PZMc) or 0.34 mmol/L sodium citrate+0.277 mmol/L inositol (PZMci, P<0.05). The blastocyst rate of added 0.5 mg/mL hyaluronic acid was significantly higher than no addition group (P<0.05). Addition of 3 mg/mL BSA-FAF blastocyst rate was significantly higher than BSA-V (41.4±4.1% vs 36.7±3.8%, P<0.05). The blastocysts formation rate and blastocysts hatch rate increased with increasing of the concentration of BSA to 5 mg/mL or added 10% FBS after 96 h cultivation, but displayed no significant difference. A higher proportion of blastocysts and cell number per blastocyst after added 50 ng/mL IGF-I to IVC medium than no addition group, but displayed no significant difference.2. Effects of activation methods on the in vitro development of porcine parthenogenetic embryos were studied. The results indicated, oocytes were first activated with 5 μM Ion for 5 min, after that, oocytes were incubated in the PZM-3 medium supplemented with 2 mmol/L 6-dimethylaminopurine (6-DMAP),5 μg/mL ctyocalasin B (CB)+10 μg/mL cycloheximide (CHX),5 μg/mL CB+2 mmol/L 6-DMAP,5 μg/mL CB+10 μg/mL CHX+2 mmol/L 6-DMAP for 4 h, respectively, the blastocyst rates were significantly higher than 5 μM Ion for 5 min then 10 μg/mL CHX for 4 h (29.7±1.1%,29.8±1.2%,30.4±1.6% and 30.2±2.7% vs 15.8±1.5%, P<0.05), but had no significant difference in blastocyst rates, ICM cell numbers, TE cell numbers and ICM/TE ratios among the four groups. When voltage was 1.25 KV/cm, the blastocyst rate of 3 pulses was significantly higher than 2 pulses (P<0.05). When pulse number was 3, the fragmentation rates of 0.80 and 1.00 KV/cm were significantly lower than 1.20 KV/cm, and blastocyst rate of 1.00 KV/cm was significantly higher than that of 0.80 and 1.20 KV/cm (P<0.05). Oocytes were first activated by electrical stimulus, after that, oocytes were incubated in the PZM-3 medium supplemented with 5 μg/mL CB+10 μg/mL CHX,10 μg/mL CHX+2 mmol/L 6-DMAP for 4 h, respectively (55.4±1.2% and 50.4±2.9%), the blastocyst rates were significantly higher than activated by electrical stimulus then 10 μg/mL CHX 2 mmol/L 6-DMAP (37.9±2.4%),5 μg/mL CB+10 μg/mL CHX+2 mmol/L 6-DMAP (36.9±3.1%) for 4 h, respectively, also the blastocyst rates were significantly higher than activated by electrical stimulus alone (40.7±1.7%) or 5 μM Ion for 5 min then 2 mmol/L 6-DMAP for 4 h (30.6±0.8%, P<0.05). The blastocyst rates of activated by electrical stimulus then 5 μg/mL CB for 4 h were significantly higher than 10 μg/mL CB for 4 h (54.0±4.1% vs 37.9±2.4%, P<0.05), but had no significant difference from 7.5 μg/ml CB, moreover 7.5 μg/ml CB group resulted in notably more ICM cell numbers and ICM/TE ratio than 5μg/ml group (P<0.05). Karyotype analysis indicated, the blastocyst cell diploid rate of activated by electrical stimulus then 7.5 μg/mL CB+10 μg/mL CHX treatment for 4 h group was significantly higher than electrical activation (84.6% vs 40.0%, P<0.05).3. Factors influencing CI-MGT were studied. The results of parthenogenetic oocytes cytoplasmic micro injection (PA-CI) indicated, there was no significant difference in blastocysts rate, ICM cell numbers, TE cell numbers and ICM/TE ratio after added 1-4 U/μL RNase inhibitor in exogenous RNA, the result indicated RNA and RNase inhibitor could be introduced into oocytes at the same time. The EGFP expression rate of 100 ng/μL EGFP-N1 DNA injecton group was significantly higher than that of 50 ng/μL and sham injection group, but blastocyst rate was significantly lower than the two groups (33.9±1.1% vs 46.4±2.1% and 50.5±1.9%, P<0.05), and there were no significant differences in blastocyst rate after injected various concentrations of Talen-BMP15 RNA, these results indicated injection of foreign DNA brought less toxicity to embryos than foreign RNA. The blastocyst rates of injected after activation 5-18 h were significantly lower than that of injected before activation and injected after activation groups. The EGFP expression rate of line EGFP-N1 DNA was significantly lower than circle (15.8±1.4% vs 11.7±1.0%, P<0.05), but displayed no significant difference in embryo development between the two groups, in addition, the blastocyst rates of foreign RNA injection groups were generally higher than DNA injection groups. When IVF zygotes were injected (IVF-CI), there was no significant difference in blastocysts rate and cell number per blastocyst after injected with spermatozoa and oocyte coincubation of 5-18 h, but EGFP expression rates of 5-6, 8-9 and 11-12 h were significantly higher than that of 14-15 and 17-18 h (12.6±0.6%,11.8±0.5% and 13.6±0.6% vs 8.7±0.8% and 6.5±1.0%, P<0.05), the result indicated that foreign DNA injected before male and female pronucleus formation (about 13 h) could result in high integration rate. The blastocyst rates of 40 and 50 ng/μL EGFP-N1 DNA injection groups were significantly higher than 100 ng/μL, but had no significant difference from 10,20,30 ng/μL and sham injection groups, and EGFP expression rates of 40,50 and 100 ng/μL were significantly higher than the other groups. When IVF zygotes were injected with Cas9 mRNA and GDF8 sgRNA, blastocyst was significantly lower than PA oocytes (13.7±0.8% vs 52.6±3.1%, P<0.05), on the contrary, the mutation efficiency of IVF embryos in the targeting site was significantly higher than PA embryos (25.0% vs 3.8%, P<0.05), the result indicated that Cas9/sgRNA system could be used for pig genome editing by injected into IVF eggs directly.4. Factors influencing ICSI-MGT were systematically studied. The results indicated, the blastocyst rates of 10 and 20 ng/μL EGFP-N1 DNA groups were significantly higher than that of 50 and 100 ng/μL (33.8±2.4% and 31.0±0.9% vs 24.4±2.7 and 16.1±0.8%, P<0.05), but EGFP expression rate of 20 ng/μL group was significantly higher than 10 ng/μL (30.5±1.5% vs 16.3±1.5, P<0.05). The blastocyst rates of spermatozoa and exogenous gene coincubation of 5,30 and 60 min were significantly higher than 24 h, but EGFP expression rates and blastocyst EGFP expression rates of 30 and 60 min were significantly higher than 5 min. There was no significant difference in embryo development rate when using different spermatozoa cryopreservation solution (DPBS, BTS and mNIM), but the blastocyst rates of various incubation buffer DPBS+EDTA (10 mmol/L EDTA) was significantly higher than DPBS (27.8±0.7% vs 22.2±1.6%, P<0.05), and mNIM (10 mmol/L EDTA+123.0 mmol/L KC1) was significantly higher than DPBS+EDTA (33.2±1.3% vs 27.8±0.7%, P<0.05), these results indicated EDTA and K+ in co-incubation buffer had positively effect on ICSI-MGT embryo development. The blastocyst rates of different sperm treatment Sonication+Frozen and NaOH were significantly higher than that of Fresh, A23187, Sonication, Frozen and Frozen three times, in addition, EGFP expression rates of Sonication+Frozen and NaOH were significantly higher than that of Fresh and A23187 treatment groups, moreover, the blastocyst rates of Sonication+Frozen and NaOH groups injected after activation were significantly higher than injected before activation (35.7±2.4% vs 30.2±0.6%; 34.5±0.3% vs 28.5+0.9%, P<0.05), the result indicated sperm treatment and activation method affected ICSI-MGT embryo development. The blastocyst rates of injected after activation 0-30 min were significantly lower than 60-90 min, but EGFP expression rate and blastocyst EGFP expression rate were significantly higher than 60-90 min, analysis of GSH level in different development time of zygotes, the GSH level in 6 h zygotes of 0-30 min group was significantly lower than 60-90 min and PA groups (3.22+0.06 vs 5.02±0.09 and 4.73+0.14, P<0.05), and there was no significant change in GSH level of 60-90 min group within 6 h and 12 h zygotes, pronucleus formation was further analyzed, the female and male pronucleus formation rate of 0-30 min injection group was significantly higher than 60-90 min (56.9+3.7% vs 36.3±2.0%, P<0.05), moreover, compact sperm of 60-90 min injection group was significantly higher than 0-30 min, these results indicated that higher parthenogenetic embryos would be leaded after prolonged the time from activation to injection. There was no significant difference in blastocyst rate after injected various DNA vectors (EGFP-N1, Anti-FMDV, PRL-EGFP and IFN-EGFP), but EGFP expression rate of line EGFP-N1 DNA was significantly higher than circle (35.9±0.2% vs 20.9±0.9%, P<0.05), and the EGFP expression rate and blastocyst EGFP expression rate of Ant-FMDV RNAi plasmid were significantly higher than the other groups (P<0.05). Cas-GDF8 RNA, Anti-FMDV and line EGFP-N1 DNA ICSI-MGT embryos were transferred into six spontaneous estrus recipient gilts, four were not re-estrus, No.7557 pregnant gilt aborted 3 fetuses, and No.7557 pregnant gilt delivered 4 piglets. Analysis the integration of EGFP and Neo gene with ear skin by PCR demonstrated that the four were transgene piglets, the result indicated that genetically modified pig offspring could be produced by optimized ICSI-MGT. |
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