Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells
Comparison of red-shifted firefly luciferase Ppy RE9 and conventional Luc2 as bioluminescence imaging reporter genes for in vivo imaging of stem cells
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DOI：
10.1117/1.jbo.17.1.016004
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发表时间：
2012-01-01
影响因子：
3.5
通讯作者：
Bulte, Jeff W. M.
中科院分区：
文献类型：
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作者：
Liang, Yajie;Walczak, Piotr;Bulte, Jeff W. M.
对移植的生物发光细胞进行无创成像的一个关键问题是，当发射波长低于600 nm时，组织中的大量光吸收被使用。具有红移光谱的荧光素酶可以潜在地绕过这一限制。我们评估并比较了萤火虫荧光素酶突变体（Ppy RE9, PRE9）与黄色荧光素酶luc2基因在细胞移植研究中的应用。采用流式细胞术对表达PRE9-Venus和luc2-Venus的C17.2神经干细胞进行分选，并在体外培养和体内移植到免疫缺陷Rag2-/-小鼠脑后进行生物发光评价。我们发现PRE9的发光稳定，在620nm处的发光峰比luc2的发光峰偏红。与luc2相比，PRE9的发射峰与ph无关，受组织吸光度的影响也比luc2小得多。然而，无论在体内还是体外，PRE9的总发射光辐射都明显低于luc2。我们得出结论，与luc2相比，PRE9在pH独立性，红移光谱，组织光穿透和信号量化方面具有良好的特性，证明进一步优化蛋白表达和酶活性是合理的。(c) 2012年中国光学仪器工程师学会（SPIE）；(jbo.17.1.016004 DOI: 10.1117/1.)
One critical issue for noninvasive imaging of transplanted bioluminescent cells is the large amount of light absorption in tissue when emission wavelengths below 600 nm are used. Luciferase with a red-shifted spectrum can potentially bypass this limitation. We assessed and compared a mutant of firefly luciferase (Ppy RE9, PRE9) against the yellow luciferase luc2 gene for use in cell transplantation studies. C17.2 neural stem cells expressing PRE9-Venus and luc2-Venus were sorted by flow cytometry and assessed for bioluminescence in vitro in culture and in vivo after transplantation into the brain of immunodeficient Rag2-/- mice. We found that the luminescence from PRE9 was stable, with a peak emission at 620 nm, shifted to the red compared to that of luc2. The emission peak for PRE9 was pH-independent, in contrast to luc2, and much less affected by tissue absorbance compared to that of luc2. However, the total emitted light radiance from PRE9 was substantially lower than that of luc2, both in vitro and in vivo. We conclude that PRE9 has favorable properties as compared to luc2 in terms of pH independence, red-shifted spectrum, tissue light penetration, and signal quantification, justifying further optimization of protein expression and enzymatic activity. (c) 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.JBO.17.1.016004]