The Termination Of ALVE1 Transcription Through CRISPR/Cas9 System And Preliminary Study For Its Function

It was stated that endogenous retroviruses are footprints left by past retroviral infections and constitute an important component of our virome.Until recently,these remnants of ancient retroviral infections were commonly presumed to form much of the so-called redundant 'junk'DNA.Recently,they are being revealed to have important physiological function in embryogenesis,embryonic stem cell development,virus infection,innate immunity and tumor development.In this study,we selected an endogenous retrovirus-avian leukosis virus subgroup E(ALVE)as a model to study its function.By using the CRISPR/Cas9 system,transcription of the endogenous retroviruses ALVE1 was successfully terminated.This study provided the foundation for further studying the function of endogenous retrovirus in the genome.A transcriptional terminator,SV40 ploy A,was knocked into the promoter region of endogenous retrovirus ALVE1 by CRISPR/Cas9 system and ALVE1 transcription was effectively terminated.This study not only successfully developed a new method to terminate endogenous retrovirus expression in poultry,but also provided a new idea to study the function of endogenous retrovirus.The main results are as follows:1.Identification and transcriptional analysis of ALVEI:Firstly,the complete sequences of ALVEI located on chicken chromosome 1 were obtained by Inverse PCR and rapid amplification of cDNA ends(RACE)technologies.The results showed that the ALVE1 sequences exist in the genome of chicken macrophage cell line HD 11 while not in the chicken fibroblast cell line DF-1.Finally,the transcription of ALVE1 in HD11 cells was identified by PCR.The results showed that the four regions of ALVE1(LTR,gag,poL and env)can be transcribed.2.Screening of transcription termination elements:In order to screen the highly efficient transcription termination sequences,the vectors containing transcription termination sequences and green fluorescent protein GFP was constructed and the termination efficiency of four transcription termination sequences were tested,including the SV40 polyA,4 × SV40 polyA,?-globin and ?-globin ? 5-7 by detecting GFP.The results showed that all of four transcription termination sequences had termination activity and the termination efficiency was over 90%.SV40 polyA was selected as the terminating element owing to its short sequences.Then,the termination efficiency of sense and antisense SV40 polyA was further tested.The results indicated that both sense and antisense SV40 polyA had transcription termination activity,but the termination activity of the antisense sequences was lower than that of the sense sequences.3.Transcription termination of ALVE1 mediated by CRISPR/Cas9 and its function study:In order to study the function of ALVE1,the homologous recombination vector containing transcription termination sequences was knocked into the promoter region of ALVE1 by CRISPR/Cas9 system and then selected by puromycin and neomycin.We obtained transgenic chicken macrophage cell line(named HD11-ALVE1-Knowdown),which had a knockdown expression of ALVEI.Next,the effect of ALVE1 termination on the cell innate immunity was further explored by RT-qPCR.Results showed that the expression of TLR3 and IFN-? were significantly decreased in HD11-ALVE1-Knowdown cells,suggesting that ALVEI may be associated with innate immunity and the avian endogenous leukemia virus may involve in host resistance to exogenous retroviruses.