Construction of the mmu_circ_0001033 lentiviral expression vector as the basis for the study of its functional experimental study in hypoxic pulmonary hypertension.

First, primers were designed to amplify the target gene fragment from the original plasmid, and recombined it into the overexpression vector after digestion through the seamless clone recognition sites contained at both ends of the primer, transferred the ligation product into the prepared bacterial sensation Then sent its monoclonal colony to a sequencing company for sequencing and identification. The looping condition was verified by running the gel and sequencing the PCR product. Finally, the constructed recombinant expression vector and packaging plasmid were used to co-transfect 293T cells, packaged the lentivirus, collected the virus stock, concentrated by ultrafiltration, and determined the titer.

The sequence of the inserted fragment in the recombinant clone was exactly the same as the sequence of the target fragment, indicating that the plasmid was constructed successfully. The PCR product was confirmed by sanger sequencing to confirm the correct sequence at the cyclarization site. By comparing the sequencing results of the constructed overexpression vector and the plasmid construction scheme, the insert sequence and the target fragment sequence in the recombinant clone are exactly as expected, which can realize the expression of the target gene after transfection of the plasmid, the PCR product running gel and sequencing results showed that mmu_circ_0001033 was successfully looped, the PCR product running gel and sequencing results showed that mmu_circ_0001033 was successfully looped. Finally, the virus titer value determined by RT-qPCR was 2.09×10⁸ TU/ml.

The mmu_circ_0001033 lentiviral expression vector was successfully constructed, which can stably transfect 293T cells and can be used for subsequent cell experiments.