دورية أكاديمية
Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis.
| العنوان: | Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis. |
|---|---|
| المؤلفون: | Zhang C; State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China., Wei ZX; State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China., Wang M; State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China., Chen YS; State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China., He ZY; State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China. |
| المصدر: | Yi chuan = Hereditas [Yi Chuan] 2022 Jul 20; Vol. 44 (7), pp. 581-590. |
| نوع المنشور: | Journal Article |
| اللغة: | English |
| بيانات الدورية: | Publisher: Ke xue chu ban she Country of Publication: China NLM ID: 9436478 Publication Model: Print Cited Medium: Print ISSN: 0253-9772 (Print) Linking ISSN: 02539772 NLM ISO Abbreviation: Yi Chuan Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Beijing : Ke xue chu ban she, |
| مواضيع طبية MeSH: | Melanoma*/genetics , Melanoma*/metabolism , Receptor, Melanocortin, Type 1*/genetics , Receptor, Melanocortin, Type 1*/metabolism, Animals ; CRISPR-Cas Systems ; Humans ; Mammals/metabolism ; Melanins/genetics ; Monophenol Monooxygenase/metabolism ; Swine |
| مستخلص: | MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color. |
| فهرسة مساهمة: | Keywords: A243T; CRISPR/Cas9; MC1R; site-directed mutagenesis Local Abstract: [Publisher, Chinese] MC1R (melanocortin 1 receptor)基因编码黑皮质素1型受体,它在配体α-黑素细胞刺激素(α-melanocyte stimulating hormone, α-MSH)的刺激下,可促进细胞内cAMP (cyclic adenosine monophosaphate)的生成,从而激活PKA (protein kinase A system)信号通路,促进黑色素合成调控的关键转录因子-小眼畸形相关转录因子(microphthalmia-associated transcription factor, MITF),以及限速酶-酪氨酸酶(tyrosinase, TYR)的表达,进而调控真黑素和褐黑素的合成,影响动物的毛色表型。有研究表明,杜洛克猪(Susscrofa domestica) MC1R蛋白第6个跨膜结构域(transmembrane domain 6, TM6)发生了A243T突变,该突变可能影响MC1R蛋白生物学功能,被认为是导致杜洛克猪红毛表型的主要因素,然而却一直未被验证过。因此,本研究以单链寡核苷酸(single- stranded oligo-deoxyribonucleotides, ssODN)作为同源重组模板,利用CRISPR/Cas9技术在人类黑色素瘤细胞系SK-MEL-2中引入MC1R基因的c.727G>A (A243T)突变,以分析其对MC1R功能的影响。结果表明ssODN发生同源重组的效率可达10%。通过对共转染ssODN和CRISPR/Cas9表达载体的细胞进行筛选和培养,获得6个单克隆细胞株,进行测序鉴定,结果未能筛选到发生预期突变的单克隆细胞株,所有单克隆细胞株皆在目标位点周围出现突变。对转染后的细胞群进行功能分析,结果发现其cAMP信号强度、MITF基因和TYR基因表达量均显著低于未转染的细胞,表明编辑MC1R可影响细胞的黑色素合成功能。本研究为深入分析MC1R突变影响动物毛色提供了进一步的参考。. |
| المشرفين على المادة: | 0 (Melanins) 0 (Receptor, Melanocortin, Type 1) EC 1.14.18.1 (Monophenol Monooxygenase) |
| تواريخ الأحداث: | Date Created: 20220720 Date Completed: 20220722 Latest Revision: 20220722 |
| رمز التحديث: | 20240628 |
| DOI: | 10.16288/j.yczz.22-037 |
| PMID: | 35858770 |
| قاعدة البيانات: | MEDLINE |
| تدمد: | 0253-9772 |
|---|---|
| DOI: | 10.16288/j.yczz.22-037 |