Construction of AIM2 Overexpression and Knockdown Lentiviral Vectors and Their Effect Verification
WANG Yu
LIU Ying
LIU Huajing
REN Feifei
WANG Yue
DIAO Bo
Abstract：Objective To construct absent in melanoma 2(AIM2)gene overexpression and knockdown lentiviral vectors,and to verify the effect of the above lentiviral overexpression and knockdown of AIM2 gene in human multiple myeloma(MM)cell line RPMI8226.Methods Recombinant plasmids containing the full AIM2 gene fragment and recom-binant plasmids containing AIM2-targeted short hairpin RNA(shRNA)gene fragments were synthesized by using recom-binant DNA technology.After validation by colony polymerase chain reaction(PCR)and sequencing,lentivirus packaging and titer determination were carried out,and RPMI8226 cells were infected with lentivirus under appropriate conditions.AIM2 overexpression experimental group were grouped:WT group(no lentivirus added,blank control),OE-NC group(50 μl OE-NC lentivirus added,negative control),OE-AIM2 group(AIM2 overexpression,66.67 μl OE-AIM2 lentivirus added).AIM2 knockdown experimental group were grouped:WT group(no lentivirus added,blank control),NC group(32.26 μl NC lentivirus added,negative control),shRNA1 group(33.33 μl shRNA1 lentivirus added,AIM2 knockdown target 1),shRNA2 group(33.33 μl shRNA2 lentivirus added,AIM2 knockdown target 2),shRNA3 group(33.33 μl shRNA3 lentivirus added,AIM2 knockdown target 3).Fluorescence was detected using fluorescence microscope,and the effects of lentivirus on the overexpression and silencing of the AIM2 gene in cells were evaluated by quantitative polymer-ase chain reaction(qPCR)and Western blot.Results The sequencing results showed that the target sequence was presented in the AIM2 overexpression recombinant plas-mids,and the 3 target sequences were contained in the AIM2 knockdown recombinant plasmids.The results of qPCR and Western blot analysis indicated that the AIM2 mRNA and protein expression levels in the OE-AIM2 group were significantly upregulated than those in the OE-NC group(all P＜0.05);the AIM2 mRNA and protein expres-sion levels in the shRNA1,shRNA2 and shRNA3 groups were all down regulated than those in the NC group(all P＜0.05).Conclusion The AIM2 gene overexpression and knockdown lentiviral vectors are successfully constructed,and the effectiveness of overexpressing and knocking down AIM2 gene is verified,resulting in RPMI826 cells with stable AIM2 overexpression and stable AIM2 knockdown.
Keywords：Multiple myelomaAbsent in melanoma 2Recombinant plasmidLentiviral vectorsEffect verifica-tion
Publication Date：2025-07-28
Online Publishing Date：2025-08-27(First online date of this platform, not the publication date of the document)
Pages：8( 565-572 )