Singer R H, Ward D C
Proc Natl Acad Sci U S A. 1982 Dec;79(23):7331-5. doi: 10.1073/pnas.79.23.7331.
The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.
鸡肌肉组织培养系统已用于原位杂交后可视化肌动蛋白基因表达。在该系统中，随着成肌细胞融合形成肌管，细胞分化在形态上是可区分的。这种分化涉及肌原纤维所需的大量肌动蛋白的产生。通过将经缺口平移掺入dUTP生物素化类似物的重组质粒进行杂交，已在乙醇和多聚甲醛保存的细胞中观察到肌动蛋白mRNA的存在。然后使用抗生物素抗体和罗丹明偶联的二抗检测生物素。或者，与罗丹明偶联的抗生物素蛋白或与生物素化过氧化物酶复合的抗生物素蛋白已用于mRNA检测。所描述的方法保留了形态细节，但与杂交条件兼容，并揭示了基因表达过程中肌动蛋白mRNA的分布情况。
