Preliminary Exploratione On The Production Of Transgenic Pig Embryos Transferred Sohlh2by Sperm Mediated Gene Transfer With Cell-Penetrating Peptides

Sohlh2is bHLH (basic helix-loop-helix) transcription factor superfamily members. It is one of the key gene primordial follicle development preparation of animal Sohlh2gene transfer has an important scientific research and application value. The method of preparing a transgenic animal, the sperm vector method which is simple to operate,less artificial mechanical damage advantages, so this method make the majority of researchers attention. However, the sperm vector method success rate is not high, the positive rate problem cannot be properly resolved, so it need to improve the foreign gene into the sperm on the basis of the efficiency of the sperm vector method, in order to achieve the purpose of improving the positive rate,and then the cell-penetrating peptides (CPPs) are a better solution. CPPs are a small peptides which be constituted by not more than30amino acid residues,and has a strong transmembrane transport capacity.In this study, we explored and compared cell-penetrating peptides (cell-penetrating peptides, CPPs) used for different cell transfection feasibility.After that, we produced the porcine transgenic expressing Sohlh2by Sperm Mediated Gene Transfer with the selected CPP. The results were as follows:1.Sohlh2gene full-length coding region sequence fragments were cloned. The size of the amplified fragment contains a gene·coding sequence for the full length is1281bp,MEGA and ORF Finder application procedures was used for bioinformatics analysis of the obtained sequences.Multiple sequence comparison showed similarity buffalo、cattle、sheep、pig、human、mouse Sohlh2gene amino acids respectively98%.96%、85%、81%and81%,Sohlh2has high conserved genes in different species at the amino acid and nucleotide levels. The secondary structure of protein analysis showed that the α-helix,β-fold,(3-angle and random coil protein constitute Sohlh2protein,protein has its own unique domain.2.Buffalo Sohlh2gene expression patterns were studied.The expression of genes in buffalo Sohlh2different tissues were detected by quantitive real time PCR (qRT-PCR).The results showed that the abundance of buffalo Sohlh2highest expression in the testis.After building lentiviral expression vector of the Sohlh2gene,and then fold dilution method was determined by packing out the virus titer which was detected virus titer of106, reached the virus-infected cells requires effective.3.Effects of CPPs on the transfection efficiency of different cells and porcine sperms were explored. Firstly, we explored the influence of C105y with Sohlh2plasmid in different incubation time,temperatures and incubated liquid on the transfection of293T cells.The results showed that C105y with Sohlh2plasmid incubated in37℃, DMEM for20min could get better transfection effect. Secondly,flow cytometry analysis of293T cells transfected by CPPs and liposomal were done.The results showed that the liposome had the highest transfection efficiency (87.72%).Transfection efficiency of C105y、Mpg、Tat、 Pep-1and Pep-3were48.61%、42.90%.8.03%、3.36%and1.98%, respectively, which were slightly lower than that of liposomes.Finally, the transfection effects of C105y,Mpg and Tat on the porcine sperm were explored,and the incubated conditions were also explored, the results showed that C105y with Sohlh2plasmid incubated in37℃, DMEM for20min could get better transfection effect. The light-emitting area of porcine sperms was observed under the laser confocal,the results showed that light-emitting area focused on post acrosome area of sperm.4.Effects of different carriers on transgenic porcine embryos were studied. Firstly, we examined the changes of sperm motility, the average velocity curve (VCL)、average linear velocity (VSL) and antegrade (STR) by treated with C105y、Mpg、Tat, lentivirus and liposomes.The results showed that, sperm motility、VCL. VSL and STR were the least affected by C105y treated, and had no significant difference with the control group(P＞0.05).After that, we produced IVF porcine embryos by using C105y and lentivirus as carriers. The blastocyst rate of C105y was higher than that of the lentivirus group (15.8±6.9vs9.1±3.5,P<0.05), and had no significance difference with the control(15.8±6.9vs13.8±5.0, P>0.05). But positive rate of C105y group was lower than of the lentivirus group(8.54±0.78vs10.38±0.95,P<0.05).The above results demonstrate that:1.We could clone buffalo Sohlh2gene with mixture cDNA of buffalo ovary and testis as template,which could use for building lentiviral vector of Sohlh2.2.Penetrating peptides C105y and Mpg incubated at37℃,DMEM for20min, which could effectively transfect Sohlh2gene plasmid into the cells and sperms,but the effects were worse than the Liposome group.3. Sperm motility、VCL、VSL and STR were the least affected by C105y treated,which showed C105y was suit for IVF carrier.4.Transgenic porcine blastocyst expressing Sohlh2were both produced by IVF with C105y and lentiviral.