Study On Preparation Of Transgenic Chicken With Sperm-mediated Gene Tradsfer

Sperm-mediated transgene technology is simple in operation and low in cost.Since Lvaitran was proposed in 1989,it has attracted extensive attention.However,the development and application of sperm vector technology are largely restricted due to the defects of sperm viability caused by co-incubation of sperm with exogenous DNA in vitro,such as difficulty in fertilization with egg cells and inability to integrate exogenous DNA into animal cell genomes.At present,the CRISPR/Cas9 system has shown a strong ability of site-directed gene editing in the experimental results of many researchers.This feature is feasible to prepare genetically modified transgenic animals with gene editing(gene mutation)by combining with sperm vector method.It is of great significance to combine the two technologies to prepare gene-edited chickens under the condition that the preparation technology of transgenic chickens is not mature at present.Therefore,this experiment aims to screen out efficient sgRNA by using Growth hormone receptor(GHR)as a model,establish incubation conditions for sperm carrier liposomes,and combine CRISPR/Cas9 system with sperm carrier technology,in the hope of establishing a simple,efficient and cheap method for preparing genetically modified chickens.The main research contents and results are as follows:Highly active sgRNA enzyme screening:in vitro experiment according to the first short small chicken growth hormone receptor gene missing part of the exon region using online sgRNA design software design 5 sgRNA sequence,and construct the front insert T7 has respectively the promoter sgRNA in vitro transcription carrier pT7-sgRNA-trac,transcription in vitro plasmid vector as a template for PCR amplification out corresponding sgRNA transcription template and purified in vitro,reuse sgRNA in vitro transcription kits,corresponding sgRNA transcription.With chicken blood genomic DNA template for PCR amplification in vitro cutting substrates and purification,according to the Cas9 in vitro enzyme kit instructions will Cas9 protein,sgRNA and cutting substrates mixed in proportion,incubation in vitro enzyme results in 1%agarose gel electrophoresis test,results:the in vitro sgRNA 1 cutting efficiency is 82.1%,sgRNA2 cutting efficiency is 57.4%,in vitro sgRNA3 cutting efficiency is 92.5%,in vitro sgRNA4 cutting efficiency is 33.7%,in vitro sgRNA5 cutting efficiency is 95.4%in vitro.DF-1 intracellular identification of sgRNA activity and sgRNA off-target analysis.sgRNA1,sgRNA3 and sgRNA5 targeted at GHR genes were selected to construct corresponding co-expression vectors of sgRNA,Cas9 and EGFP.Pmd19t-neo-zyb plasmid vectors of left and right homologous arms were constructed,and Neo R expression structure was connected with left and right homologous arms to construct target target vector PMD19t-neo-zyb.Will sgRNA,Cas9 protein and EGFP expression vectors respectively and targeting vector transfection cells,DF-1 by G418 screening,pick monoclonal cell proliferation and culture,all the positive monoclonal cell genomic DNA by PCR amplification appraisal NeoR fixed-point inserted,and insert the appraisal not fixed-point monoclonal cell genome by PCR amplification GHR gene detects the target gene sequence and sequencing identification by gumming point whether editor,including 16 sgRNA1 seedlings for fixed-point inserted into the positive cell clones have 2,editing efficiency is 12.5%;Of the 23 cell clones obtained by sgRNA3,9 were positive for site-specific insertion and 2 were non-site-specific insertion,but the target sequence was mutated,and the editing efficiency was 47.8%.Among the 24 cell clones obtained from sgRNA5,11 were positive for site-insertion and 2 were unsite-insertion but the target sequence was mutated,with an editing efficiency of 54.2%.According to online miss analysis software design sgRNA1,sgRNA3,sgRNA5 choose five miss potential sites respectively,and design the corresponding primers,to filter all the Neo R resistance as a template for positive cell clones genome PCR amplification corresponding potential miss loci from sequence and sequencing:five miss potential sites respectively chosen sgRNA happened miss not detected.Sperm incubation conditions established:freshly collected the cock semen,sperm,take 200 μL with incubation liquid wash detergent,liquid detergent incubation liquid diluent,DMEM medium respectively,1640 medium and PBS buffer,incubation 10 min、30 min、60 min、90 min,observe the sperm vitality to choose the appropriate incubation fluid,can choose to maintain higher sperm vitality of incubation fluid respectively with pX330 plasmid incubation for 10 min,30 min,60 min,90 min,sperm motility and observation in semi-quantitative PCR identification ingest exogenous DNA screening optimal incubation time.The other group of experiments was to use 0 μL,1 μL,1.5 μL and 2 μL liposomes to encase plasmid DNA before incubation.After the incubation bath time according to the optimal time established in the previous section,then semi-quantitative PCR was used to identify the uptake of exogenous DNA and the proportion of viable sperm to select the appropriate liposome volume.The fertilization rates of different incubation methods were compared by artificial insemination,and the number of embryos of fertilized eggs with exogenous plasmids was identified by PCR to determine the best incubation method.Results different incubation liquid after incubation,M199 culture as incubation fluid,the incubation of energy after the sperm percentage is 78.48%,and diluent for incubation liquid vitality of the sperm percentage is 18.26%,DMEM medium have sperm vitality for incubation liquid proportion is 19.28%,1640 medium for incubation fluid has vitality sperm percentage is 20.44%,PBS buffer for incubation fluid has vitality sperm percentage is 21.57%;The optimal incubation time was when the incubation time was 60 min,and there was no significant difference between the proportion of viable sperm and the incubation time of 10 min and 30 min(P>0.05).After the incubation time exceeded 60 min,the ability of sperm to absorb exogenous DNA was no longer significantly increased(P>0.05).Liposome incubation the best dosage of liposomes 1.5 μL,the proportion of energy under the condition of sperm and without liposome has no obvious difference(p>0.05),liposome more than 1.5 μL ingest exogenous DNA ability no longer increased with the increase of liposome volume increased significantly(p>0.05),when the liposome reached 2(including L,vigorous sperm less and the proportion of liposome group also had significantly decreased(p<0.05);The difference was not significant(P>0.05).The difference was 51.18%in conventional incubation and 44.78%in liposome incubation(P<0.05).The difference was 14.94%in simple incubation of exogenous DNA and 28.89%in liposome incubation of exogenous DNA(P<0.05).Editing embryo or individual identification:DF-1 cells were screened for highly efficient co-expression vectors of sgRNA,Cas9 protein and EGFP,and sperm were co-incubated with liposomes under the established appropriate incubation conditions,artificial insemination,detection of embryo disc level at stage X and tissue level of newly hatched chicks gene editing:Detection 1087 First stage blastoderms X,identified three fertilized eggs gene editing,editing efficiency was 0.28%,the preliminary estimate genetically editor 3 genes in fertilized egg cell number respectively the first X period the proportion of the total number of cells a preliminary estimate of 10.4%,12.5%,10.4%,the chicken embryo sex,PCR identification of genomic DNA of gene editing three fertilized eggs are female,speculated that the CRISPR/Cas9 system gene editing period before the zygote to 8 cell stage;The chick tissue has not been identified as a sample for gene editing,and the causes may be varied.This test by screening highly targeted GHR gene sgRNA,establish suitable for chicken sperm carrier incubation conditions,combining the two,in the first stage embryo dish in the fixed-point gene editing,X for small chicken preparation to set up a new technical method,as well as other gene editing and even gene targeting of genetically modified(gm)provides a new thought and method of preparation of chicken.