| Intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) is one of the most popular approaches for generating transgenic animal. In employment of ICSI-MGT, the fertilization can be completed without considering of its vitality and a large DNA fragment can be transferred into oocytes with a relative high transgenic efficiency in comparison with IVF-SMGT. Thus, ICSI-MGT has been widely used for production of transgenic animals. In this study, factors influencing the transgenic efficiency of ICSI-MGT and mechanism of exogenous DNA binding to sperm were investigated, and the feasibility of generating transgenic rabbits expressing n-3 fatty acid desaturase gene (Fat-1) by ICSI-MGT was also explored.1. Effects of rabbit sperm pretreating methods on ICSI-MGT transgenic efficiency were explored. There was no significant difference in the sperm DNA damage between thawing after long preservation in liquid nitrogen and thawing at once after frozen without cryoprotection (P>0.05), and in the rate of two pronuclears formation (71.4%vs 67.5% and 77.2%), oocytes cleaved (76.7% vs 79.5% and 80.4%) and blastocysts developed (39.7% vs 42.0% and 40.2%) among ICSI-MGT oocytes using the long frozen, frozen-thawed at once and fresh sperm (P>0.05). As to the transgenic efficiency, the rate of EGFP-positive embryos and EGFP-positive blastocysts in the group of long fronzen sperm were significantly higher than that of the fresh sperm group (43.8% vs 18.6%; 56.1% vs 12.8%, P<0.05), but it was not significantly different from the frozen-thawed at once group (43.8% vs 40.2%; 56.1% vs 46.8%, P>0.05). Furthermore, the effects of the different sources of sperm on ICSI-MGT transgenic efficiency were evaluated and found that epididymal sperm had a extremely higher rate in binding exogenous plasmid than that of ejaculated sperm regardless of before or after DNasel digestion (before:77.5% vs 17.0%; after:47.6% vs 5.2%, P<0.01), moreover, the sperm from epididymis produced more EGFP-positive embryos and EGFP-positive blastocysts after ICSI-MGT compared to ejaculated sperm group (32.4% vs 10.8%; 40.2% vs 9.2%, P<0.05). The results obtained from agarose gel electrophoresis of seminal plasma and exogenous plasmid incubated complexes revealed that the exogenous plasmid gradually appeared degradation, and the degradation degree was increased as the concentration of seminal plasma increased, but the degradation was inhibited by addition of EDTA into the incubation system. At the same time, addition of EDTA to the sperm/DNA incubattion system for ICSI-MGT could significantly increase the rate of EGFP-positive embryos and EGFP-positive blastocysts (25.5% vs 10.8%; 36.0%vs 9.2%, P<0.05). These results indicated that (1) rabbit sperm long-frozen in liquid nitrogen without cryoprotection can be used for producing transgenic embryos by ICSI-MGT; (2) the deoxyribonuclease in the rabbit seminal plasma can degrade the exogenous DNA, and this action can be inhibited by EDTA, which will increase the ICSI-MGT transgenic efficiency.2. Effects of activation and culture methods on the in vitro development of rabbit ICSI-MGT embryos were investigated. The cleavage rate of oocytes activated with 5 μM Ion for 5 min and then cultured in 2 mM 6-DMAP or 5 μg/mL CHX+2 mM 6-DMAP for 1.5 h was significantly higher than that of oocytes activated with 5 μM Ion for 5 min and then cultured 5 μg/mL CHX for 1.5 h and oocytes without activation. Moreover, more oocytes activated with 5 μM Ion for 5 min and then cultured in 2 mM 6-DMAP or 5 μg/mL CHX+2 mM 6-DMAP for 1.5 h developed blastocysts (41.3%) and resulted in grade I embryo (65.4%) in comparison with other three groups (P<0.05). The blastocyst development rate and blastocyst cell number of grade Ⅰ embryos were significantly higher than the grade Ⅱ and grade Ⅲ embryos (P<0.05) and similar to the in vivo embryos (P>0.05). When culture medium was supplemented with different concentration (0,10,50,100,250 and 500 nM) of 1-phosphate-sphingosine (SIP), an apoptosis inhibitor, addition of 50 nM and 100 nM SIP resulted in higher blastocysts rate and improved the quality of blastocysts (P<0.05). The proportion of embryos with normal chromosomes (2n = 44) was also higher when they were cultured in the medium supplemented with 100 nM SIP in comparison with embryos cultured without SIP (94.7%vs 78.6%, P<0.05). Moreover, when the grade Ⅱ embryos were cultured in the medium supplemented with 100 nM SIP, the actin microfilament distributed regularly on the region of cell junction and inner cell mass. While the actin microfilament distributed irregularly and loosely on the region of cell junction and inner cell mass in embryos cultured without SIP. These results suggeste that activation of oocytes with 5 μM Ion for 5min and then cultured in 5 μg/mL CHX +2 mM 6-DMAP for 1.5 h is suitable for producing rabbit ICSI-MGT embryos. Supplemented culture medium with 100 nM SIP can improve the embryo quality by reducing the degree of fragmentation, maintaining the normal ploidy of chromosomes and distribution of actin microfilament in embryos.3. Factors (plasmid concentration and ratio of linear-to-circular plasmid) influencing the ICSI-MGT transgenic efficiency were evaluated, and transgenic rabbit expressing Fat-1 gene was generated. The capacity of sperm uptaking exogenous DNA and the rate of EGFP-positive embryos after ICSI-MGT trended to increase as the DNA concentration increased. However, as the concentration was increased to 500 ng/μL, the cleavage rate and blastocyst yield were significantly decreased in comparison with other groups. In the 30 ng/μL group, the blastocyst development rate was significantly higher than the 50 ng/μL group (49.0% vs 33.3%, P<0.05), and the EGFP-positive embryos and EGFP-positive blastocysts were significantly higher than the 10 ng/μL and 15 ng/μL groups (56.3% vs 45.6% and 21.2%,55.3% vs 39.0%and 33.3%, P<0.05). The result of agarose gel electrophoresis of genome extracted from sperm incubated with different concentration of exogenous DNA revealed that degradation of sperm genome increased with the increase of exogenous DNA concentration, and then the developmental competence of embryos decreased. When the ratio of linear-to-circular plasmid was fixed at 1:1, significantly more exogenous DNA binded to sperm and higher transgenic efficiency was achieved in comparison with other ratios (1:0,1:4 and 0:1) (P<0.05). A total of 281 EGFP-positive embryos at 2-8 cells stage were transplanted into ten estrus synchronized recipients and oocyte donor rabbits, two of them became pregnant and given birth to seven pups (No.1-7). The integration of the Fat-1 gene was confirmed in six out of seven live pups by PCR and Southern blot. The expression of the Fat-1 gene in these six pups was also detected by RT-PCR. The expression of EGFP protein was confirmed by confocal microscopy and Western Blot analysis in all six RT-PCR-positive pups. The Gas Chromatography-Mass Spectrometry (GC-MS) analysis showed that n-3 polyunsaturated fatty acids were increased in muscle tissue of transgenic rabbits, while n-6 polyunsaturated fatty acids were significantly decreased. The ratio of n-6/n-3 in the muscle (0.79 ± 0.1,1.0 ± 0.1,1.9 ± 0.1,0.94 ± 0.1,0.7 ± 0.1 and 0.6 ± 0.1) of transgenic rabbits was 15-fold lower than the wild-type rabbit (17.5 ± 1.4). These results demonstrate that 30 ng/μL of exogenous DNA and 1:1 of linear-to-circular plasmid is appropriate for ICSI-MGT. Fat-1 transgenic rabbits can be produced by ICSI-MGT and display low ratio of n-6/n-3 PUFAs.4. To investigate the molecular mechanism of sperm and exogenous DNA interaction, Native-PAGE electrophosis was used for separating complexes of DNA fragment/sperm proteins and free sperm proteins. A distinct band was found after coomassie blue staining and seven proteins were obtained by mass spectrometry. Analysis of the physical/chemical properties and conservaed region of the seven proteins found that the protein IAM38 (sperm inner acrosomal membrane protein IAM38) was conform to the feature of the DBPs. Moreover, only protein IAM38 presents a series of identify conserved domains with IG and DNA-binding structure including zinc finger, leucine zipper and helix-loop-helix after analysing by online software of SMART. Furthermore, it was verified by Western Blotting that the CD4 exist in sperm but no in seminal plasma. Blocking CD4 through CD4 monoclonal antibody decreased the DNA-uptaking capacity of sperm (7.5% vs 51.4%, P<0.01), but did not influence the DNA-binding capacity of sperm (68.3% vs 75.7%, P>0.05). Moreover, the EGFP-positive embryos and EGFP-positive blastocysts were also decreased in comparison with the control group (7.8%vs 43.8%; 7.1%vs 56.1%, P<0.01). These results confirm that foreign DNA first binds to the transmembrane IAM38 of sperm plasma membrane and then forms the complex of DNA/IAM38/CD4 with CD4 to complete the transportation of exogenous DNA into the nucleus of sperm. |
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