{"id":2867,"date":"2025-03-07T10:45:41","date_gmt":"2025-03-07T09:45:41","guid":{"rendered":"https:\/\/welkerlab.hu\/?page_id=2867"},"modified":"2026-04-16T12:29:45","modified_gmt":"2026-04-16T10:29:45","slug":"gfxfp","status":"publish","type":"page","link":"https:\/\/welkerlab.hu\/en\/gfxfp\/","title":{"rendered":"GFxFP"},"content":{"rendered":"
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GFxFP reporter systems<\/h2>\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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\n\t\t\t\t\t\t\t\t\tSchematics of the principle of the GFxFP reporter assay. The sequences of the two GFP halves are indicated in green. Each of them contains both a non-overlapping segment (green), and an overlapping segment (green with grey lines). The expression cassette is driven by a CAG promoter (white arrow) and terminated by a SV40 (Simian Vacuolating Virus 40) polyA signal (pA, grey box). Expression from the first GFP half is terminated by an early STOP codon (black mark), and thus, results in no detectable green fluorescence. The second GFP half has neither Kozak sequence nor ATG. Upon nuclease cleavage at the target site (blue box) the generated double-strand DNA break can be repaired by HDR of the overlapping homologous sequences. Thus, an intact GFP sequence is created from which a functional fluorescent GFP (green) is transcribed.\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t
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Example: Comparison of cleavage efficiencies of different LbCas12a variants on the same target with different PAM sequences in the plasmid based GFxFP assay in mouse N2a cells.<\/strong>
Percentages of GFP positive cells counted above the background level, resulting from the action of various nucleases. Error bars show the mean \u00b1 standard deviation of percentages measured in 3 independent transfections.<\/p>\t\t\t\t\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t\t\t\t

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Transfection details<\/strong>
Cells were seeded onto 48-well plates a day before transfection at a density of 3\u00d7104 cells\/well. The next day, at around 40% confluence, cells were transfected with plasmid constructs using Turbofect reagent.), briefly as follows: 250 ng total plasmid DNA (2 ng GFxFP plasmid, 124 ng crRNA and nuclease expression plasmid, and 124 ng mCherry expression plasmid to monitor the transfection efficiency) and 1 \u03bcL Turbofect were mixed in 50 \u03bcL serum free DMEM and the mixture was incubated for 30 min at room temperature prior being added to cells. Three parallel transfections were made from each sample. Cells were analysed by flow cytometry two days post-transfection. The EGFP signal was analysed in the mCherry positive population. Background fluorescence was determined by using a crRNA-less, inactive LbCas12a nuclease expression vector as negative control and this background value was subtracted from each sample. Three parallel transfections were made for each case.<\/p>

Addgene number #89052<\/a>
Cloning site to accomodate the target sequence is between BamHI and EcoRI sites.<\/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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References<\/strong>
T\u00f3th, Eszter ; Weinhardt, N\u00f3ra ; Bencsura, Petra ; Husz\u00e1r, Krisztina ; Kulcs\u00e1r, P\u00e9ter Istv\u00e1n ; T\u00e1las, Andr\u00e1s ; Fodor, Elfrieda ; Welker, Ervin
Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells<\/em>
BIOLOGY DIRECT 11 Paper: 46 , 14 p. (2016)<\/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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T\u00f3th, Eszter; Czene, Bernadett C; Kulcs\u00e1r, P\u00e9ter Istv\u00e1n; Krausz, Sarah Laura; T\u00e1las, Andr\u00e1s; Nyeste, Antal; Varga, \u00c9va; Husz\u00e1r, Krisztina; Weinhardt, N\u00f3ra; Ligeti, Zolt\u00e1n; Borsy, Adrienn \u00c9va; Fodor, Elfrieda; Welker, Ervin
Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants<\/em>
NUCLEIC ACIDS RESEARCH 46 : 19 pp. 10272-10285. , 14 p. (2018)<\/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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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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GFxFP riporter rendszer A GFxFP riporter rendszer Cas nukle\u00e1zok has\u00edt\u00e1si hat\u00e9konys\u00e1g\u00e1nak meghat\u00e1roz\u00e1s\u00e1ra \u00e9s \u00f6sszehasonl\u00edt\u00e1s\u00e1ra alkalmas. A GFxFP riporter rendszer el\u0151nye, hogy genomi kontextust\u00f3l f\u00fcggetlen\u00fcl vizsg\u00e1lhat\u00f3 benne az egyes targetek has\u00edt\u00e1si hat\u00e9konys\u00e1ga. Alatta bal oldalt \u00e1bra, jobb oldal\u00e1n a magyar\u00e1zat: „A GFxFP riportervizsg\u00e1lat elv\u00e9nek v\u00e1zlata. A k\u00e9t GFP f\u00e9l szekvenci\u00e1ja z\u00f6lddel van jel\u00f6lve. Mindegyik tartalmaz egy […]<\/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-2867","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages\/2867","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=2867"}],"version-history":[{"count":19,"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages\/2867\/revisions"}],"predecessor-version":[{"id":3185,"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/pages\/2867\/revisions\/3185"}],"wp:attachment":[{"href":"https:\/\/welkerlab.hu\/en\/wp-json\/wp\/v2\/media?parent=2867"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}