{"id":2787,"date":"2025-01-27T10:16:58","date_gmt":"2025-01-27T09:16:58","guid":{"rendered":"https:\/\/welkerlab.hu\/?page_id=2787"},"modified":"2026-04-16T12:32:56","modified_gmt":"2026-04-16T10:32:56","slug":"hek293-clover-sejtvonal","status":"publish","type":"page","link":"https:\/\/welkerlab.hu\/en\/hek293-clover-sejtvonal\/","title":{"rendered":"HEK293.clover
cell line"},"content":{"rendered":"
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HEK293.clover
cell line<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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The HEK293.clover cell line was designed to test the cleavage efficiency of Cas12a nucleases with the TTTN PAM sequence by a large target disruption assay. HEK293.clover cells were generated as follows: a modified clover sequence was inserted after the ATG of human prion protein coding sequence with a self-cleaving donor plasmid by NHEJ-knock-in in HEK293 cells (293 H, Gibco).<\/p>

Modified clover sequence<\/strong>
Three consecutive thymidines or adenines are marked by yellow or blue, respectively.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Disruption assay<\/strong>
Cells were plated one day prior to transfection on 48-well plates at a density of approximately 25,000-30,000 cells\/well. Cells were co-transfected with two types of plasmids: Cas12a variant expression plasmid (137 ng) and crRNA and mCherry coding plasmid (97 ng) using 1 \u00b5L Turbofect reagent per well in 48-well plates. Transfected cells were analysed six days post-transfection by flow cytometry. For control an inactive LbCas12a expressing nuclease vector was used. Disruption activities of the nucleases were calculated as follows:
1 \u2013 (sample GFP % \/ average GFP % of negative controls)
Transfection efficiency was calculated via mCherry expressing cells. Transfections were performed in triplicate.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Reference<\/strong>
T\u00f3th, Eszter ; Varga, \u00c9va ; Kulcs\u00e1r, P\u00e9ter Istv\u00e1n ; Kocsis-Jutka, Vir\u00e1g ; Krausz, Sarah Laura ; Nyeste, Antal ; Welker, Zsombor ; Husz\u00e1r, Krisztina ; Ligeti, Zolt\u00e1n ; T\u00e1las, Andr\u00e1s ; Welker, Ervin
Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases<\/em>
NUCLEIC ACIDS RESEARCH 48 : 7 pp. 3722-3733. , 12 p. (2020)<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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HEK293.clover sejtvonal A HEK293.clover sejtvonalat arra hoztuk l\u00e9tre, hogy a TTTN PAM szekvenci\u00e1val rendelkez\u0151 Cas12a nukle\u00e1zok has\u00edt\u00e1si hat\u00e9konys\u00e1g\u00e1t is tudjuk vizsg\u00e1lni nagysz\u00e1m\u00fa targeten disruption assay-vel. A m\u00f3dos\u00edtott clover szekvencia a hum\u00e1n prion feh\u00e9rje ATG-je ut\u00e1n ker\u00fclt beilleszt\u00e9sre a HEK293 sejtek (293 H, Gibco) genomj\u00e1ba egy \u00f6nhas\u00edt\u00f3 donor plazmiddal, NHEJ-knock-in seg\u00edts\u00e9g\u00e9vel. M\u00f3dos\u00edtott clover szekvenciaH\u00e1rom egym\u00e1st k\u00f6vet\u0151 […]<\/p>","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"elementor_header_footer","meta":{"site-sidebar-layout":"no-sidebar","site-content-layout":"page-builder","ast-site-content-layout":"full-width-container","site-content-style":"unboxed","site-sidebar-style":"unboxed","ast-global-header-display":"","ast-banner-title-visibility":"","ast-main-header-display":"","ast-hfb-above-header-display":"","ast-hfb-below-header-display":"","ast-hfb-mobile-header-display":"","site-post-title":"","ast-breadcrumbs-content":"","ast-featured-img":"disabled","footer-sml-layout":"","theme-transparent-header-meta":"","adv-header-id-meta":"","stick-header-meta":"","header-above-stick-meta":"","header-main-stick-meta":"","header-below-stick-meta":"","astra-migrate-meta-layouts":"set","ast-page-background-enabled":"default","ast-page-background-meta":{"desktop":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"ast-content-background-meta":{"desktop":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"tablet":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""},"mobile":{"background-color":"var(--ast-global-color-5)","background-image":"","background-repeat":"repeat","background-position":"center center","background-size":"auto","background-attachment":"scroll","background-type":"","background-media":"","overlay-type":"","overlay-color":"","overlay-gradient":""}},"footnotes":""},"class_list":["post-2787","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages\/2787","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/comments?post=2787"}],"version-history":[{"count":16,"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages\/2787\/revisions"}],"predecessor-version":[{"id":3191,"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages\/2787\/revisions\/3191"}],"wp:attachment":[{"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/media?parent=2787"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}