Preliminary Research Of A Depressed Marmoset Model Mediated By CRISPR/Cas9 Gene Modification

Objective CRISPR/Cas9 technology was used to edit the depression susceptibility genes of marmosets at the cellular and embryonic levels,providing a basis for the establishnent of depression models or susceptibility models modified by genes in marmosets;Horseradish peroxidase(HRP)labeled antibody was prepared to provide resources for the immunological detection system of marmosets.Methods(1)Cultured skin,kidney,liver,heart and lung tissue cells of marmosets in vitro(2)Verification at the cellular level:10 sgRNA of depression susceptibility genes SIRT1,SLC6A4,LHPP,HTR1A and MTHFR,marmoset kidney cells transfected with Cas9/ABE/CBE plasmid.After resistance screening,the genome was extracted.PCR amplified target fragment,sequencing,and T7E1 enzyme digestion fragnent decting mutation rate.(3)Verification of embryo level:the progesterone content in the serum of marmosets was detected by ELISA to detect the menstrual cycle;rhFSH+hCG is used for superovulation;Oocytes were collected by follicular puncture;IVM was used to obtain mature oocytes;Rectal electrical stimulation/penile concussion technique is used of collect sperm;IVF is used to obtain fertilized eggs;Four sgRNAs with high knockout efficiency were selected for zygote injection,genome extraction,amplification and sequencing.(4)Purification and identification of serum IgG by affinity chromatography and SDS-PAGE electrophoresis;Immunodouble diffusion method was used to determine the antiserum titer,and modified sodium periodate labeling method was used to prepare IgG-HRP labeled antibody against marmoset.The working concentration and specificity of IgG-HRP labeled antibody against marmoset were determined by ELISA and western-blot.Results(1)Establishment of the kidney cell culture,transfection,and resistance screening system of marmoset:the biological properties of F2-F5 generation cells were stable,which could be used in the following cell experiments.The suitable concentration of puromycin 4?g/ml and pyrimidin 8?g/ml were selected for cell resistance.(2)CRISPR/Cas9-mediated validation of marmoset cell level was effective:sequencing results showed that spurious peaks began to appear near the PAM sequence of sgRNA,and mutations were introduced in all 10 sgRNA.Mononucleotide replacement ABE has mutations at three sites with a very low mutation rate<10%.CBE has no mutation.CRISPR/Cas9 with high gene editing efficiency was selected as the gene editing technology for the construction of a gene modified depression model.(3)Results of the menstrual cycle of marmosets:the menstrual cycle of marmosets was about 27.68±4.76d,of which the follicular stage was about 9.56±2.95 d,and the luteal stage was about 18.12±3.96d.The follicular period is short,and it is suitable to do the experiment from March to November.(4)Sperm collection technique:two sperm collection techniques were compared,and high sperm activity rectal electrical stimulation technique was used.(5)The validation of the CRISPR/Cas9 technology-mediated embryo level in marmoset was effective:sgRNA and Cas9 protein were injected together into the prokaryote of zygote.Except that LHPP 1 did not introduce mutations into sgRNA,the mutation rates of MTHFR,HTR1A and SLC6A4 genes were about 41.6%,30%and 41.6%,respectively.The CRISPR/Cas9 technology successfully introduced mutant embryos,accounting for 75%of the total number of embryos tested.(6)Serum IgG 95%;antiserum titer 1:64;the reference concentrations of ELISA and western-blot were 1:25 6000 and 1:15 000,respectively.Conclusion The kiney cell culture,transfection and screening system of marmoset was established.CRISPR7Cas9 mediated the editing efficiency of 5 depression susceptibility genes at the cell level.We demonstrated that CRISPR/Cas9 technology can be applied to edit marmoset embryos.This study improved the assisted reproductive technology of marmosets convincing experimental data,and the achieved preliminary validation of CRISPR/Cas9 technology at the level of cells and embryos.IgG-HRP labeled antibody was prepared,which provided resources for the immunological detection system of marmoset pathogen and molecular immunological detection system.