RESUMO
BACKGROUND: Many human genetic diseases arise from point mutations. These genetic diseases can theoretically be corrected through gene therapy. However, gene therapy in clinical application is still far from mature. Nearly half of the pathogenic single-nucleotide polymorphisms (SNPs) are caused by G:C>A:T or T:A>C:G base changes and the ideal approaches to correct these mutations are base editing. These CRISPR-Cas9-mediated base editing does not leave any footprint in genome and does not require donor DNA sequences for homologous recombination. These base editing methods have been successfully applied to cultured mammalian cells with high precision and efficiency, but BE4 has not been confirmed in mice. Animal models are important for dissecting pathogenic mechanism of human genetic diseases and testing of base correction efficacy in vivo. Cytidine base editor BE4 is a newly developed version of cytidine base editing system that converts cytidine (C) to uridine (U). RESULTS: In this study, BE4 system was tested in cells to inactivate GFP gene and in mice to introduce single-base substitution that would lead to a stop codon in tyrosinase gene. High percentage albino coat-colored mice were obtained from black coat-colored donor zygotes after pronuclei microinjection. Sequencing results showed that expected base changes were obtained with high precision and efficiency (56.25%). There are no off-targeting events identified in predicted potential off-target sites. CONCLUSIONS: Results confirm BE4 system can work in vivo with high precision and efficacy, and has great potentials in clinic to repair human genetic mutations.
Assuntos
Animais , Camundongos , Adenosina Desaminase , Citosina , Sistemas CRISPR-Cas , Edição de Genes/métodos , Sequência de Bases , Western Blotting , Modelos Animais , Reação em Cadeia da Polimerase em Tempo Real , MutaçãoRESUMO
Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45ß and transfection expression of c-Fos and GADD45ß in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45ß can reverse the induced cell death. Co-transfection of both c-Fos and GADD45ß into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45ß. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45ß can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45ß/siRNA-GADD45ß did not affect each other's expression. Moreover, analysis of c-Fos and GADD45ß regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45ß mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.
Assuntos
Antígenos de Diferenciação/genética , Apoptose/efeitos dos fármacos , Benzopiranos/farmacologia , Biomarcadores Tumorais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/genética , Neoplasias da Bexiga Urinária/patologia , Proliferação de Células , Sobrevivência Celular , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Transdução de Sinais , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genéticaRESUMO
Crude brazilin extract from Sappan wood has demonstrated strong anti tumor activity in the mouse model of human bladder carcinoma and clinical trial for intravesical therapy. Purified brazilin was confirmed the most active molecule in inhibition of bladder carcinoma T24 cells. Brazilin decreased proliferation and viability of T24 cells in a dose- and time-dependent manner, with a calculated LC50 of 32 µg/mL. More than 1,000 of genes were found upregulated and down regulated by brazilin treatment in digital gene expression profiling. Gene ontology analysis indicated that stress response, apoptosis, and cell cycle regulatory pathways were highly enriched. Among the regulated genes, c-Fos was the most and specifically upregulated. Overexpression of c-Fos in T24 cells resulted in tumor cell specific changes in cell morphology and viability. Over expression of stress-responsive gene, HSP70, and other highly upregulated genes did not have any effect on cell growth. Brazilin may inhibit T24 cell growth and trigger cell death through a c-Fos-mediated and tumor cell specific signaling pathway. Further studies of its down stream mediators may help to identify better tumor cell type specific drug targets.