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Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation.
Kolb, Alexander L; Reynoso, Marinaliz; Matheny, Ronald W.
Afiliação
  • Kolb AL; Military Performance Division, US Army Research Institute of Environmental Medicine, 10 General Greene Ave., Building 42, Natick, MA 01760, USA.
  • Reynoso M; Military Performance Division, US Army Research Institute of Environmental Medicine, 10 General Greene Ave., Building 42, Natick, MA 01760, USA.
  • Matheny RW; Military Operational Medicine Research Program, 810 Schreider St./MRDC-RTO/B201, Ft. Detrick, MD 21702, USA.
J Biol Methods ; 8(3): e151, 2021.
Article em En | MEDLINE | ID: mdl-34514012
Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability to genetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: J Biol Methods Ano de publicação: 2021 Tipo de documento: Article

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Idioma: En Revista: J Biol Methods Ano de publicação: 2021 Tipo de documento: Article