Construction And Functional Analysis Of The Common Allelic Gene Double-knockout Vector System

Gene targeting by homologous recombination involves the introduction of exogenous homologous sequences, as part of so called of targeting vector and their recognition by the cellular machinery. Currently, there has been no targeting system can achieve a null allele though one targeting cycle, sequencing targeting always a laborious work. This study offered a biallelic knockout system, which may make all these easier.The system is composed of vectors of pGT-V1 and pGT-V2, which is quite similar to each other except the positive markers. To facilitate their construction, firstly we created two bicistronic eukaryotic expression vector pIRES-Neo-GFP and pIRES-Hyg-RED. To construct the pBS246/MCS/TK plasmid, two Multiple Cloning Sites (MCS) composed of carring a series of rare 8 bases pair restriction enzyme cutting sites were sequentially instead into pBS246, outside of two equidirectional LoxP fragment, then a Herpes Simplex Thymidine Kinase (HSV-TK) gene cassette, which was excised from pORF-HSV1tk plasmid, was cloned between the MCSs. Finally, combine the pBS246/MCS/TK plasmid with the Neo-IRES-GFP and Hyg-IRES-RED fragments respectively yielding the targeting vectors of vectors of pGT-V1 and pGT-V2 for different floxd positive selectable markers. Fluorescence microscope, flow cytometry and antibiotics were conducted and the results proved the two vectors are functional. In order to further confirm their capability of knockout two allele and standard the targeting process, mice MSTN gene was successfully cloned into the vectors generating the MSTN targeting system. Simultaneously, Mock PCR was performed and the targeting checking system was well established.All in all, in this work, the common allelic gene double-knockout vector was successfully created and targeting system for the mice myostatin gene was well prepared for the coming study.