Browsing by Subject "CRISPR/Cas9"

Now showing 1 - 3 of 3
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    CRISPR-mediated WNK4 point mutation aggravates tumor progression and weakens chemotherapy sensitivity in gastric cancer
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2025) Ying, Xiaojun; Ying, Zhen; Gao, Xiaobing; Wang, Yong; Lv, Xinting
    Objective. Gastric cancer (GC) is the fifth most common malignancy, the molecular targets of which have been increasingly explored in recent years. As a serine/threonine protein kinase, the role of WNK lysine deficient protein kinase 4 (WNK4) in GC was clarified in this study. Methods. Human GC lines AGS and MKN45 were stably transfected with a WNK4 mutant constructed by the CRISPR/Cas9 method and treated with cis-dichlorodiammine platinum (CDDP, 2 μg/mL) and 5-fluorouracil (5-FU, 5 μg/mL) for 48h. Tumor-bearing mice were established with 5×106 mutant-type AGS cells, and injected with 40 mg/kg WP1066, the inhibitor of signal transducer and activator of transcription 3 (STAT3), for 21 days. Cell malignant potential and tumor growth were assessed. STAT3 activation was identified by western blot and immunohistochemistry. The interaction between WNK4 and STAT3 was determined using co-immunoprecipitation and immunofluorescence co-localization. Results. WNK4 mutation promoted proliferation and invasion, and upregulated the p-STAT3/STAT3 value in GC cells with/without 5-FU and CDDP treatments, while inhibiting apoptosis of GC cells without drug treatment. In tumor-bearing mice, WNK4 mutation accelerated tumor growth, increased levels of p-STAT3, STAT3, and p-STAT3/STAT3, and strengthened the co-immunoprecipitation and co-localizing with STAT3; however, these effects were reversed by WP1066 treatment. Conclusion. Through activating STAT3, WNK4 mutation impacts both the natural and drug-treated growth of GC cells or tumors, suggesting a new avenue for preclinical research.
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    Melanocyte stem cells in skin diseases and their potential in cell-based therapy
    (Universidad de Murcia, Departamento de Biologia Celular e Histiologia, 2022) Wang, Zi Han; Liu, Li-Ping; Zheng, Yun-Wen
    Melanocytes have a complex function and play an important role in a variety of regulatory mechanisms in the human system. Melanocyte stem cells (MelSCs) serve as a reservoir to replenish the melanocytes by regenerating new ones, and they are capable of self-renewal and differentiation to maintain their homeostasis, repair, and regeneration in tissues. The numerical decrease and functional impairment of MelSCs may be closely related to the development and treatment response of many skin diseases. However, the current knowledge about MelSCs mainly comes from studies in mice, and little is known about human MelSC markers; especially, their markers are still unclear or lack consensus. This leads to uncertainty in clinical findings, which further limits our comprehensive understanding of pigmentary disorders and also hinders the progress of new treatments. Thus, in this review article, combined with our previous and current work, we summarize and update the recent advances in MelSC research, including the molecular markers of human MelSCs and their niche, as well as the association of MelSCs with skin diseases, including vitiligo, hair greying, and melanoma. Due to the limited tools available to explore the identified characteristics of human MelSCs, pluripotent stem cells can provide a new research model for further study, especially combined with CRISPR/Cas9 technology. The visualization of human MelSCs’ development and differentiation can help to identify their molecular characteristics and understand their cellular fate dynamically, which will allow us not only to further explore their roles in associated diseases, but also to achieve MelSC-based cellular therapy.
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    Production of genetically modified porcine embryos by lipofection of zona pellucida-intact oocytes using the CRISPR/Cas9 system
    (2023-01-18) Piñeiro Silva, Celia; Navarro Serna, S.; Belda Perez, R.; Gadea Mateos, J. J.; Fisiología
    The generation of genetically modified pigs has an important impact thanks its applications in basic research, biomedicine, and meat production. Cloning was the first technique used for this production, although easier and cheaper methods were developed, such as the microinjection, electroporation, or lipofection of oocytes and zygotes. In this study, we analyzed the production of genetically modified embryos via lipofection of zona-pellucida-intact oocytes using LipofectamineTM CRISPRMAXTM Cas9 in comparison with the electroporation method. Two factors were evaluated: (i) the increment in the concentration of the lipofectamine–ribonucleoprotein complexes (LRNPC) (5% vs. 10%) and (ii) the concentration of ribonucleoprotein within the complexes (1xRNP vs. 2xRNP). We found that the increment in the concentration of the LRNPC had a detrimental effect on embryo development and a subsequent effect on the number of mutant embryos. The 5% group had a similar mutant blastocyst rate to the electroporation method (5.52% and 6.38%, respectively, p > 0.05). The increment in the concentration of the ribonucleoprotein inside the complexes had no effect on the blastocyst rate and mutation rate, with the mutant blastocyst rate being similar in both the 1xRNP and 2xRNP lipofection groups and the electroporation group (1.75%, 3.60%, and 3.57%, respectively, p > 0.05). Here, we showed that it is possible to produce knock-out embryos via lipofection of zona-pellucida-intact porcine oocytes with similar efficiencies as with electroporation, although more optimization is needed, mainly in terms of the use of more efficient vesicles for encapsulation with different compositions.