Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Cheung, Sylvia T.; Shakibakho, Soroush; So, Eva; Mui, Alice

doi:10.3791/52807-v

Cited by 2 publications

(2 citation statements)

References 9 publications

(10 reference statements)

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“…Commercially available transfection reagents (cationic lipid and polyamine based) have been reported for transfecting plasmid DNA in RAW 264.7 cells. They have focused not only on the transfection rate but also on the toxicity limit of the transfection reagent [49].…”

Section: Cytotoxicity Assaymentioning

confidence: 99%

RGDK-lipopeptide for targeting genetic vaccines to antigen presenting cells

Rahaman,

Chaudhuri

2023

Biomed. Mater.

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To ensure effective immune response in genetic immunizations, DNA/mRNA vaccines need to be delivered to body’s antigen presenting cells (APCs) which is a challenging task. This is primarily due to presence of high concentrations of various degradative enzymes inside them. To this end, mannose receptor (over expressed in APCs) selective cationic liposomes have been used in the past for delivering antigen-encoded plasmid DNA to APCs. APCs also express integrin receptors on their cell surfaces. However, studies aimed at delivering DNA vaccines into APCs via integrin receptors have not yet been undertaken. Herein, we report on the use of cationic liposomes of a priorly disclosed α5β1 integrin receptor selective RGDK-lipopeptide for macrophage transfection. In this study, we have used pCMV-GFP (as model DNA vaccine) and RAW 264.7 cells (mouse macrophages cells) as model APC. We show that the liposomes of RGDK-lipopeptide containing a previously reported endosome-disrupting histidinylated lipid and DOPE (as co-lipid) in 0.5:0.5:1.0 mole ratio are the most competent in transfecting macrophage cells (44%). Findings in the fluorescence resonance energy transfer based membrane fusogencity assay revealed that the enhanced macrophage transfection efficiency of the liposomes containing RGDK-lipopeptide, endosome-disrupting histidinylated and DOPE may originate from its higher membrane fusogenicity than that for liposomes containing only RGDK-lipopeptide and DOPE. The presently described biologically safe liposomal formulations of RGDK-lipopeptide are expected to find biomedical applications in future for combating cancer and infectious diseases through genetic immunizations.

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Section: Cytotoxicity Assaymentioning

confidence: 99%

RGDK-lipopeptide for targeting genetic vaccines to antigen presenting cells

Rahaman,

Chaudhuri

2023

Biomed. Mater.

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“…Since MHC-II functions primarily in antigen-presenting cells, we examined whether FBXO11 could also negatively regulate MHC-II expression in immune cells. Mouse macrophage–derived RAW264.7 was used in the experiments given its relatively high transfection efficiency ( 36 , 37 ). The overexpression of FBXO11 suppressed the protein level of MHC-II, but not MHC-I, as confirmed in nonimmune cells, and the inhibition was not conferred by the catalytically inactive ΔF mutant ( Fig.…”

Section: Resultsmentioning

confidence: 99%

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA

Kasuga

Ouda

Watanabe

et al. 2023

Proc. Natl. Acad. Sci. U.S.A.

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Major histocompatibility complex (MHC) class I and II molecules play critical roles in the activation and regulation of adaptive immunity through antigen presentation to CD8+ and CD4+ T cells, respectively. Strict regulation of MHC expression is critical for proper immune responses. CIITA (MHC class II transactivator), an NLR (nucleotide-binding domain, leucine-rich-repeat containing) protein, is a master regulator of MHC class II (MHC-II) gene transcription. Although it has been known that CIITA activity is regulated at the transcriptional and protein levels, the mechanism to determine CIITA protein level has not been elucidated. Here, we show that FBXO11 is a bona fide E3 ligase of CIITA and regulates CIITA protein level through ubiquitination-mediated degradation. A nonbiased proteomic approach for CIITA-binding protein identified FBXO11, a member of the Skp1–Cullin-1–F-box E3 ligase complex, as a binding partner of CIITA but not MHC class I transactivator, NLRC5. The cycloheximide chase assay showed that the half-life of CIITA is mainly regulated by FBXO11 via the ubiquitin–proteasome system. The expression of FBXO11 led to the reduced MHC-II at the promoter activity level, transcriptional level, and surface expression level through downregulation of CIITA. Moreover, human and mouse FBXO11 –deficient cells display increased levels of MHC-II and related genes. In normal and cancer tissues, FBXO11 expression level is negatively correlated with MHC-II. Interestingly, the expression of FBXO11 , along with CIITA , is associated with prognosis of cancer patients. Therefore, FBXO11 is a critical regulator to determine the level of MHC-II, and its expression may serve as a biomarker for cancer.

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Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Cited by 2 publications

References 9 publications

RGDK-lipopeptide for targeting genetic vaccines to antigen presenting cells

RGDK-lipopeptide for targeting genetic vaccines to antigen presenting cells

FBXO11 constitutes a major negative regulator of MHC class II through ubiquitin-dependent proteasomal degradation of CIITA

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