{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,9,9]],"date-time":"2024-09-09T07:40:41Z","timestamp":1725867641307},"reference-count":0,"publisher":"Journal of Biological Methods","issue":"3","license":[{"start":{"date-parts":[[2021,7,15]],"date-time":"2021-07-15T00:00:00Z","timestamp":1626307200000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JBM"],"abstract":"<jats:p>Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability togenetically 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 (NF&amp;kappa;B) 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. NF&amp;kappa;B 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 (TNF&amp;alpha;) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P &amp;le; 0.01 for both). LPS did not stimulate TNF&amp;alpha; 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.<\/jats:p>","DOI":"10.14440\/jbm.2021.359","type":"journal-article","created":{"date-parts":[[2021,7,16]],"date-time":"2021-07-16T19:15:40Z","timestamp":1626462940000},"page":"1","source":"Crossref","is-referenced-by-count":1,"title":["Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation"],"prefix":"10.14440","volume":"8","author":[{"given":"Alexander","family":"L. Kolb","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marinaliz","family":"Reynoso","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ronald","family":"W. Matheny Jr","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"5574","published-online":{"date-parts":[[2021,7,15]]},"container-title":["Journal of Biological Methods"],"original-title":[],"link":[{"URL":"https:\/\/polscientific.com\/jbm\/index.php\/jbm\/article\/download\/359\/417","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/web-api.polscientific.com\/uploads\/file\/asp\/202404251116058267b2131.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/web-api.polscientific.com\/uploads\/file\/asp\/202404251116058267b2131.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,9,9]],"date-time":"2024-09-09T06:30:10Z","timestamp":1725863410000},"score":1,"resource":{"primary":{"URL":"https:\/\/polscientific.com\/journal\/JBM\/8\/3\/10.14440\/jbm.2021.359"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,15]]},"references-count":0,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2021,9,2]]}},"URL":"https:\/\/doi.org\/10.14440\/jbm.2021.359","relation":{},"ISSN":["2326-9901"],"issn-type":[{"type":"electronic","value":"2326-9901"}],"subject":[],"published":{"date-parts":[[2021,7,15]]}}}