RESUMO
Programmable DNA nucleases such as TALENs and CRISPR/Cas9 are emerging as powerful tools for genome editing. Dual-fluorescent surrogate systems have been demonstrated by several studies to recapitulate DNA nuclease activity and enrich for genetically edited cells. In this study, we created a single-strand annealing-directed, dual-fluorescent surrogate reporter system, referred to as C-Check. We opted for the Golden Gate Cloning strategy to simplify C-Check construction. To demonstrate the utility of the C-Check system, we used the C-Check in combination with TALENs or CRISPR/Cas9 in different scenarios of gene editing experiments. First, we disrupted the endogenous pIAPP gene (3.0 % efficiency) by C-Check-validated TALENs in primary porcine fibroblasts (PPFs). Next, we achieved gene-editing efficiencies of 9.0-20.3 and 4.9 % when performing single- and double-gene targeting (MAPT and SORL1), respectively, in PPFs using C-Check-validated CRISPR/Cas9 vectors. Third, fluorescent tagging of endogenous genes (MYH6 and COL2A1, up to 10.0 % frequency) was achieved in human fibroblasts with C-Check-validated CRISPR/Cas9 vectors. We further demonstrated that the C-Check system could be applied to enrich for IGF1R null HEK293T cells and CBX5 null MCF-7 cells with frequencies of nearly 100.0 and 86.9 %, respectively. Most importantly, we further showed that the C-Check system is compatible with multiplexing and for studying CRISPR/Cas9 sgRNA specificity. The C-Check system may serve as an alternative dual-fluorescent surrogate tool for measuring DNA nuclease activity and enrichment of gene-edited cells, and may thereby aid in streamlining programmable DNA nuclease-mediated genome editing and biological research.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Animais , Células Cultivadas , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Fluorescência , Técnicas de Inativação de Genes/métodos , Genes Reporter , Vetores Genéticos/genética , Células HEK293 , Recombinação Homóloga , Humanos , Células MCF-7 , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Suínos , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/genética , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismoRESUMO
IL-1 plays a major role in inflammation and autoimmunity through activation of nuclear factor kappa B (NFkappaB) and MAPKs. Although a great deal is known about the mechanism of activation of NFkappaB and MAPKs by IL-1, much less is known about the down-regulation of this pathway. Suppressor of cytokine signaling (SOCS)-3 was shown to inhibit IL-1-induced transcription and activation of NFkappaB and the MAPKs JNK and p38, but the mechanism is unknown. We show here that SOCS-3 inhibits NFkappaB-dependent transcription induced by overexpression of the upstream IL-1 signaling molecules MyD88, IL-1R-activated kinase 1, TNF receptor-associated factor (TRAF)6, and TGFbeta-activated kinase (TAK)1, but not when the MAP3K MAPK/ERK kinase kinase-1 is used instead of TAK1, indicating that the target for SOCS-3 is the TRAF6/TAK1 signaling complex. By coimmunoprecipitation, it was shown that SOCS-3 inhibited the association between TRAF6 and TAK1 and that SOCS-3 coimmunoprecipitated with TAK1 and TRAF6. Furthermore, SOCS-3 inhibited the IL-1-induced catalytic activity of TAK1. Because ubiquitination of TRAF6 is required for activation of TAK1, we analyzed the role of SOCS-3 on TRAF6 ubiquitination and found that SOCS-3 inhibited ubiquitin modification of TRAF6. These results indicate that SOCS-3 inhibits IL-1 signal transduction by inhibiting ubiquitination of TRAF6, thus preventing association and activation of TAK1.
Assuntos
Interleucina-1/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Dosagem de Genes , Humanos , Complexos Multiproteicos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Fosforilação , Ligação Proteica , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas , Ubiquitina/metabolismoRESUMO
The Reg3 peptides INGAP-PP and human Reg3α/ß (HIP) have been hypothesized to stimulate ß-cell neogenesis in the pancreas. Administration of INGAP-PP has been shown to cause an increase in ß-cell mass in multiple animal models, reverse streptozotocin (STZ) induced diabetes in mice and reduces HbA1c levels in type 2 diabetic humans. In this study, we have examined the ability of the INGAP-PP and HIP peptides to induce ß-cell formation in vivo in normal mice through short-term administration of the peptides. We assessed the peptides ability to induce an increase in extra-islet insulin-positive cell clusters by looking at ß-cell number by point counting morphometry on pancreata that had been randomized using the smooth fractionator principle in non-diabetic NMRI mice after short-term injections of the peptides (5 d). Five day continuous BrdU labeling was used to determine if the new ß-cells were derived from replicating ß-cells. Real time quantitative RT-PCR and immuno-histochemistry was used to analyze changes in pancreatic transcription factor expression. A 1.5- to 2-fold increase in the volume of small extra-islet insulin-positive clusters post 5 d treatment with INGAP-PP and HIP as compared with mice treated with a non-peptide control or scrambled peptide (p<0.05) (n = 7) was found. Five day continuous BrdU infusion during the 5 d period showed little or no incorporation in islets or small insulin clusters. Five days of treatment with INGAP-PP or HIP, showed a tendency toward increased levels of pancreatic progenitor markers such as Ngn3, Nkx6.1, Sox9 and Ins. These are the first studies to compare and indicate that the human Reg3 α/ß (HIP) peptide has similar bioactivity in vivo as INGAP by causing formation of small ß-cell clusters in extra-islet pancreatic tissue after only 5 d of treatment. Upregulation of pancreatic transcription factors may be part of the mechanism of action.