[Construction and application of PGRN and Rev-erbβ double genes knockout HEK293 cell lines]

Area of Science:

• Molecular Biology
• Gene Regulation
• CRISPR/Cas9 Gene Editing

Background:

• The interaction between Progranulin (PGRN) and Rev-erbβ is not fully understood at the molecular level.
• Previous studies have not elucidated the physiological significance of PGRN's role in Rev-erbβ-mediated gene transcription.
• There is a need to investigate how PGRN affects the transcriptional activity regulated by Rev-erbβ.

Purpose of the Study:

• To investigate the molecular mechanism and physiological significance of the PGRN-Rev-erbβ interaction.
• To generate and characterize a HEK293 cell line with double knockout of Rev-erbβ and PGRN genes.
• To determine the effect of PGRN on Rev-erbβ's regulation of target gene promoter transcriptional activity.

Main Methods:

• CRISPR/Cas9 gene editing was employed to create a Rev-erbβ and PGRN double knockout HEK293 cell line (C3-6/23).
• Lentiviral vectors carrying Cas9 and specific sgRNAs targeting the PGRN gene were constructed and utilized for cell line generation.
• Quantitative reverse transcription PCR (qRT-PCR) and Western blotting were used to confirm PGRN knockout, and genetic complementation was performed.

Main Results:

• Successfully generated a monoclonal HEK293 cell line with double knockout of Rev-erbβ and PGRN (C3-6/23), confirmed by deletion mutagenesis and lack of detectable PGRN mRNA and protein.
• Demonstrated that PGRN influences Rev-erbβ's regulation of target gene promoter transcriptional activity.
• Observed an enhancement in Rev-erbβ's transcriptional regulation of target genes in the presence of PGRN.

Conclusions:

• The CRISPR/Cas9 system effectively created a Rev-erbβ and PGRN double knockout cell line.
• PGRN plays a role in modulating Rev-erbβ's regulation of target gene promoter transcription.
• Further research is required to fully elucidate the mechanism by which PGRN mediates Rev-erbβ's transcriptional regulation.