A Positive-negative Selection Cassette For BAC Recombination And Its Application In ZFN Mediated Knock-in

Traditional gene cloning techniques require PCR,restriction enzyme digestion,ligation technologies and so on.Not only the processes are cumbersome,but also theyare limited by of PCR conditions and restriction sites,and may leave a lot of redundant fragments in the counstruct.Seamless cloning technology can overcome the shortcomings of traditional gene cloning technologies.We have developed a seamless cloning technology which based on RED recombination.Take advantage of this technology for the recombination of BAC clones,we have constructed a donor plasmid used for knock-in of eGFP targeting TMEM18 gene using zinc finger nucleases.RED recombination system is derived from the lambda phage recombination system,which contains three recombination essential genes,such as exo?bet and gam.pKD46 plasmid is a homologous recombination supporting plasmid,In addition to containing the above-mentioned three kinds of genes expcressing recombinant necessary proteins by the control of arabinose promoter paraB,pKD46 also contains a temperature-sensitive replication origin orR101.In this paper,the pKD46 plasmid was used to mediate RED recombinant.In order to facilitate the recombination process,the donor DNA usually contains a resistance selection gene.We constructed a positive/negative selection cassette,pheS-kana cassette,in which kana resistant gene is used for positive selection and Ala294Gly mutant pheS for negative selection.These two genes are fused under the control of a promoter of a E.coli gene NEOKAN to form a positive/negative selection cassette.PheS gene encodes the alpha subunit of Phe aminoacyl,tRNA synthetases.At first,this gene was cloned in a T vector and then mutant at the 294 site from alanine to glycine by plasmid mutation PCR.This mutation resulted in its substrate binding space to be expanded and acceptable for para-chlorophenylalanine(p-Cl-Phe),so that p-Cl-Phe will be introduced into the protein and was toxic to E.coli cells,and at the end,resulting in cells death.We have tested the tolerance of the E.coli cells containing the mutant PheS clones to p-Cl-Phe.The result showed that the mutant PheS cells could resist against Kanamycin(25?g/ml),and E.coli cells DH5a cannot grow.when the concentration of p-Cl-Phe reach at 15 mM,These results suggested that the pheS-kana cassette system has the function of double selections.Based on these results,we used pKD46 to mediate BAC clone recombination by RED recombination,and to establish a eGFP donor plasmid for TMEM18 gene knock-in.The first step,NEOKAN-pheS-kana cassette was recombined into the the target site of TMEM18 gene in BAC clone RP23-25C13 by RED recombination utilizing kana resistant to select positive clones.We transformed pKD46 plasmid into the BAC clone.We used 100 mM L-arabinose to induce expression of recombinant proteins and then made competent cells.A NEOKAN-pheS-kana fragment was synthetized by PCR,which flanked with 50bp arms homologous to TMEM18 at both ends.After enzyme digestion by DpnI?electrophoresis and purification,the PCR product was transformed into DH5a competent cells which containing these recombinant proteins,the cells were spread on Kana(25?g/ml)/chloramphenicol(12.5?g/ml)LB plate,and incubated in 30 ? for 24 hours,then single clones were selected,amplified and identified by PCR..The second step,eGFP fragment was recombined into BAC clone to replace the NEOKAN-pheS-kana cassette.We used the same method that described above to obtain BAC competent cells containing Recombinant proteins.A eGFP fragment which flanked with 50 bp arms homologous to TMEM18 was synthesized by PCR,it was purified and transformed into the BAC competent cells containing the recombinant protein.The cells were spread on 15 mM of p-Cl-Phe/chloramphenicol(12.5?g/ml)LB plate and incubated at 30 ? for 24 hours,then signal clones were selected,and the eGFP-BAC clones were identified by PCR..The third step,we sub-cloned the eGFP flanked with 1-2 kb of two arms homologous to TMEM18,into a mini BRR322 vector by RED recombinant technology.the eGFP-BAC competent cells containing recombinant proteins were manufactured with the same method that described above.A mini BRR322 vector fragment,which contains the basic plasmid replicon?ampicillin-resistance gene and 50 bp arms homologous to TMEM18 with Nco I?EcoR I restriction sites at both ends,was synthesized by PCR,it was purified and transformed into BAC competent cells containing the recombinant protein.The cells were spread on the ampicillin(100?g/ml)LB plate,and incubated at 30 ? for 24 hours,then the single clones were selected and identified by Nco I?EcoR I digest.Finally,The donor plasmid pBRR3-eGFP was generated for eGFP knock-in targeting TMEM18 gene.We co-transfected mouse myoblast C2C12 cells with the donor plasmid pBRR3-eGFP and a ZFN plasmid which was constructed in our lab for targeting TMEM18 in.Cell fluorescence was observed after transfection for 48h.The results demonstrated that zinc finger nucleases successfully cut TMEM18 and induced eGFP knock-in.In summary,the pheS-kana positive/negative selection cassette can be effectively used in RED recombination of BAC clones.We build a new technology for RED recombination in vitro using our positive/negative selection system.Using this technology,we have established a eGFP donor plasmid for TMEM18 knock-in.Based on this technology,a eGFP knock-in cells targeting TMEM18 were established.It laid the foundation for further researches in the use of the RED recombination and the function of TMEM18.