Plasmid Construction for Gene Expression

The Hsp70 and RB1 promoters were cloned by PCR from the genome of PC12 cells, extracted with a Genomic DNA Kit (Tiangen Biotech., China). The plasmids Hsp70-pro(ΔpolyA⁺) and Hsp70-pro(ΔpolyA⁻) were constructed by replacing the CMV promoters of pEGFP-N1 and pEGFP-N1 (ΔpolyA⁻) with the Hsp70 promoter, using the restriction endonucleases AseI and EcoRI. The Hsp70 and RB1 ORFs were cloned from PC12 cDNA obtained by RT-PCR. The Hsp70 and RB1 ORFs were inserted into Hsp70-pro(ΔpolyA⁺) using BamHI to generate the Hsp70-pro-Hsp70(ΔpolyA⁺) and Hsp70-pro-Rb(ΔpolyA⁺) plasmids. RB1-pro(ΔpolyA⁺), RB1-pro(ΔpolyA⁻), RB1-pro-Hsp70(ΔpolyA⁺), RB1-pro-Rb(ΔpolyA⁺) and RB1-pro-Hsp70(ΔpolyA⁻) were constructed in a similar way. Cloning primers were as follows: Hsp70 promoter sense: 5′-TGGGAGAGGAGAGTGTGTCG-3′, antisense: 5′- GGGCGGAGAAGATCTCGAAG-3′; RB1 promoter sense: 5′-TCTTTGTAGCTGGACCTGGGCCT-3′; antisense: 5′-GGGAGCCAGCGAGCTGTGGAG-3′; Hsp70 ORF sense: 5′-ATGTCGGTGGTGGGCATAGAC-3′; antisense: 5′-ATCAATGTCCATCTCAGGAAGC-3′; RB1 ORF sense: 5′-ATGCCGCCCAAAACCCCCCGAAAAACGGCC-3′; antisense: 5′-AGCATGGATACCTCAAACAAGGAAGAGAAA-3′12 (link). 293T cells were transfected with all types of plasmids and sorted using a BD AriaIII (Becton Dickinson, USA).