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Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines.

ABSTRACT: Cell lines recapitulating physiological processes can represent alternatives to animal or human studies. The 3T3-L1 cell line is used to mimic adipocyte function and differentiation. Since transfection of 3T3-L1 cells is difficult, we used a modified 3T3-L1 cell line overexpressing Cas9 for a straightforward generation of gene knock-outs. As an example, we intended to generate 3T3-L1 cell lines deficient for adhesion G protein-coupled receptors Gpr64/Adgr2 and Gpr126/Adgr6 using the CRISPR/Cas approach. Surprisingly, all the generated knock-out as well as scramble control cell lines were unresponsive to isoprenaline in respect to adiponectin secretion and lipolysis in contrast to the wild type 3T3-L1 cells. We, therefore, analysed the properties of these stable Cas9-overexpressing 3T3-L1 cells. We demonstrate that this commercially available cell line exhibits dysfunction in cAMP signalling pathways as well as reduced insulin sensitivity independent of gRNA transfection. We tried transient transfection of plasmids harbouring Cas9 as well as direct introduction of the Cas9 protein as alternate approaches to the stable expression of this enzyme. We find that transfection of the Cas9 protein is not only feasible but also does not impair adipogenesis and, therefore, represents a preferable alternative to achieve genetic knock-out.

SUBMITTER: Suchy T

PROVIDER: S-EPMC8735834 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAdipose tissue is a critical regulator of lipid storage and endocrine function. Impairment of the recruitment of new adipocytes in the adipose tissue is associated with ectopic fat accumulation, diabetes and insulin resistance. Torreya nucifera, an evergreen conifer that grows in warm temperate climates, has been found to exert beneficial effects against inflammation, infection and diabetes. However, the molecular mechanisms responsible for these effects at the cellular level remain unknown. This study aimed to investigate effects of Torreya nucifera seed oil (TNSO) on 3T3-L1 adipocyte differentiation and its underlying regulatory mechanism.MethodsTo investigate the effects of TNSO on adipocyte differentiation, 3T3-L1 cells were induced to differentiate for 5 days in the presence of 0.75 μL/mL TNSO. Oil Red O staining and an assay for intracellular triglyceride were performed to determine the extent of lipid accumulation in 3T3-L1 cells. To elucidate the underlying mechanism of TNSO, adipogenic gene expression was analyzed using quantitative real-time PCR. Moreover, we monitored TNSO-derived activation of PPARγ and STAT3 with 3T3-L1 reporter cell lines engineered to secrete Gaussia luciferase upon the interaction of a transcription factor to its DNA binding element.ResultsOil Red O staining revealed that TNSO improved the differentiation of 3T3-L1 preadipocytes into mature adipocytes. The mRNA levels of adipogenic genes, including adiponectin, fatty acid synthase (FAS) and adipocyte fatty acid-binding protein (FABP4), were upregulated and intracellular triglyceride levels increased upon TNSO treatment. We also established that adipocyte differentiation was improved by TNSO-derived activation of PPARγ and STAT3.ConclusionsOur results suggest that TNSO improves adipocyte differentiation by regulating the activation of adipogenic transcription factors, indicating that it may serve as a potential treatment strategy for adipocyte dysfunction.

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Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic knock-out adipocyte cell lines.

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