Gene overexpression involves cloning the coding sequence (CDS) of a target gene into a suitable plasmid or viral vector. By utilizing regulatory elements built into the vector backbone, the gene is transcribed and translated in large quantities under artificially controlled conditions, thereby achieving overexpression of the target gene. In addition, reporter genes can be selected for tracking, or resistance genes for screening.

    Overexpression technology can be used to screen for new gene functions and drug targets. Commonly used viral vectors include adeno-associated virus (AAV) vectors, lentiviral vectors, adenoviral vectors, and retroviral vectors, which are widely applied in in vitro and in vivo gene function studies.

I. Experimental Procedures for Gene Overexpression Using DNA Recombination Technology

1. Amplify the target gene: Perform an LR recombination reaction between a vector containing the target gene and a lentiviral vector. Use an 8 μL reaction mixture containing 100 ng of pENTR 3c dual, 1 μL of pLenti6.3/V5-DEST, 2 μL of LR recombinase mixture, and TE buffer added to a total volume of 8 μL. Incubate overnight at 25°C in a water bath for 12–16 hours. Then, add 1 μL of the recombinant plasmid from the overnight incubation to 10 μL of TOP10 competent cells for transformation, followed by colony picking and shaking the culture.

2. Recombination and ligation of the target gene with the eukaryotic expression vector: Extract the plasmid and perform restriction enzyme digestion to verify that the target gene has been accurately inserted. Digest the target fragment using a 20 μL digestion mixture; subject the digested gene fragment to agarose gel electrophoresis, and recover the gel containing the target gene; ligate the recovered target gene with pcDNA 4.0. Combine pcDNA 4.0, the target gene, and the ligase, and incubate at 16°C for 10 h; add all ligation products to 100 μL of Top10 competent cells for transformation; verify the resulting products via agarose gel electrophoresis after restriction enzyme digestion.

3. Sequence Analysis: Isolate positive clones, extract the target gene, and perform an NCBI BLAST search to verify the sequence and rule out gene mutations that may have occurred during recombination.

4. Cell Transfection: Transfect 293T cells with the successfully recombined eukaryotic expression plasmid pcDNA4.0-14-3-3θ. Seed 2 × 10² 293T cells in a 6 cm dish the night before; perform transfection using 15 μL of Lip2000, 6 μL of the recombinant eukaryotic expression plasmid, and serum-free culture medium; after 6 hours, replace the medium with serum-containing medium (10% serum); collect the protein after 72 hours and verify high expression of the target protein via Western blot.

5. Screening and validation of cell lines overexpressing the target protein: Transfect cells with the recombinant plasmid containing the target gene and pcDNA4.0-cherry, respectively. The transfection system and conditions were the same as those described above. 72 hours after transfection, screening was performed using culture medium containing 100 μg/mL zeocin. Three weeks later, proteins were extracted from the resulting cell clones, and Western blot analysis was performed to verify high-level expression of the target protein; RNA was extracted, and real-time RT-PCR was performed to verify high-level expression of the target protein at the mRNA level.

II. CRISPRa Gene Overexpression Technology

2.1 Technical Principle

    The CRISPRa system targets specific promoter or enhancer regions by linking a transcriptional activation domain between the sgRNA and the Cas9 protein, thereby upregulating gene transcriptional activity and activating gene expression.

2.2 Key Components

    The CRISPRa system primarily consists of a specific sgRNA and a Cas9-transcription activation domain fusion protein. The transcription activation domain is typically VP64 or p65AD, linked to the C-terminus of Cas9 via a linker. sgRNA design requires selection of a promoter or enhancer region of the target gene. The CRISPRa method is generally suitable for large genes that cannot be directly overexpressed exogenously.

2.3 Experimental Procedures

(1) Construct Cas9-activation domain fusion expression vectors and sgRNA expression vectors;

(2) Transfect the expression vectors into cells to achieve intracellular expression of CRISPRa;

(3) Extract RNA and use qPCR to assess changes in the mRNA expression levels of the target gene to confirm gene activation.

(4) If the mRNA levels of the target gene are significantly upregulated compared to the control group, the experiment is considered successful.

III. Comparison of CRISPRa with Traditional ORF Overexpression Techniques

IV. Characteristics and Applications of the Three Types of Overexpression Vectors

    KMD Bioscience has launched a service for establishing stable cell lines with overexpression. With extensive experience in establishing stable cell lines—including, but not limited to, 4T1, 786-O, 9607, THP-1, MCF7/ADR, H1299, 2F-2B, A549, V79, CHO, and HepG2—we can help you successfully screen for monoclonal cell lines that stably express your target gene.

    This article is intended as a reference for science enthusiasts. It is not a substitute for professional expertise or practical experimental procedures that require more detailed and specialized information. If any content infringes on copyright, please contact the author to have the disputed material removed immediately.