Department of Biological Sciences, Columbia University, Howard Hughes Medical Institute, New York, NY 10027, USA.
Genetics. 2024 Oct 7;228(2). doi: 10.1093/genetics/iyae126.
One problem that has hampered the use of red fluorescent proteins in the fast-developing nematode Caenorhabditis elegans has been the substantial time delay in maturation of several generations of red fluorophores. The recently described mScarlet-I3 protein has properties that may overcome this limitation. We compare here the brightness and onset of expression of CRISPR/Cas9 genome-engineered mScarlet, mScarlet3, mScarlet-I3, and GFP reporter knock-ins. Comparing the onset and brightness of expression of reporter alleles of C. elegans golg-4, encoding a broadly expressed Golgi resident protein, we found that the onset of detection of mScarlet-I3 in the embryo is several hours earlier than older versions of mScarlet and comparable to GFP. These findings were further supported by comparing mScarlet-I3 and GFP reporter alleles for pks-1, a gene expressed in the CAN neuron and cells of the alimentary system, as well as reporter alleles for the pan-neuronal, nuclear marker unc-75. Hence, the relative properties of mScarlet-I3 and GFP do not depend on cellular or subcellular context. In all cases, mScarlet-I3 reporters also show improved signal-to-noise ratio compared to GFP.
一个阻碍红色荧光蛋白在快速发展的秀丽隐杆线虫（Caenorhabditis elegans）中应用的问题是，几代红色荧光团的成熟存在相当大的时间延迟。最近描述的 mScarlet-I3 蛋白具有可能克服这一限制的特性。我们在这里比较了 CRISPR/Cas9 基因组工程改造的 mScarlet、mScarlet3、mScarlet-I3 和 GFP 报告基因敲入的亮度和表达起始时间。比较了广泛表达的高尔基体驻留蛋白编码基因 golg-4 的报告基因等位基因的表达起始时间和亮度，我们发现 mScarlet-I3 在胚胎中的检测起始时间比旧版 mScarlet 早几个小时，与 GFP 相当。这些发现通过比较在 CAN 神经元和消化系统细胞中表达的基因 pks-1 的 mScarlet-I3 和 GFP 报告基因等位基因以及用于全神经元、核标记 unc-75 的报告基因等位基因得到进一步支持。因此，mScarlet-I3 和 GFP 的相对性质不依赖于细胞或亚细胞环境。在所有情况下，mScarlet-I3 报告基因与 GFP 相比，信号噪声比也得到了改善。
