Nellemann C, Abell K, Nørremølle A, Løkkegaard T, Naver B, Röpke C, Rygaard J, Sørensen S A, Hasholt L
Department of Medical Biochemistry and Genetics, Panum Institute, University of Copenhagen, Denmark.
Mol Cell Neurosci. 2000 Oct;16(4):313-23. doi: 10.1006/mcne.2000.0872.
The Huntington disease gone encodes the protein huntington, which is widely expressed during embryonic development and in mature tissues. In order to elucidate the physiological function of huntington, which so far is unknown, we intend to study the effect of antisense down-regulated huntington expression. We have found an inhibiting effect of a phosphorothioated oligodeoxynucleotide (PS-ODN) added to the culture medium of embryonic teratocarcinoma cells (NT2) and postmitotic neurons (NT2N neurons) differentiated from the NT2 cells. Specific inhibition of expression of endogenous huntington was achieved in NT2N neurons in the concentration range of 1-5 microM PS-ODN, whereas no inhibition was obtained in NT2 cells. We describe in detail the selection of the target sequence for the antisense oligo and the uptake, intracellular distribution, and stability of the antisense PS-ODN in the two cell types. Antisense down-regulation of huntington in this model of human neurons represents a suitable approach to study its normal function.
亨廷顿病基因编码亨廷顿蛋白，该蛋白在胚胎发育过程中和成熟组织中广泛表达。为了阐明目前尚不清楚的亨廷顿蛋白的生理功能，我们打算研究反义下调亨廷顿蛋白表达的效果。我们发现，添加到胚胎性畸胎瘤细胞（NT2）培养基以及由NT2细胞分化而来的有丝分裂后神经元（NT2N神经元）中的硫代磷酸化寡脱氧核苷酸（PS-ODN）具有抑制作用。在1 - 5 microM PS-ODN浓度范围内，NT2N神经元中内源性亨廷顿蛋白的表达受到特异性抑制，而在NT2细胞中未观察到抑制作用。我们详细描述了反义寡核苷酸靶序列的选择以及反义PS-ODN在这两种细胞类型中的摄取、细胞内分布和稳定性。在这种人类神经元模型中对亨廷顿蛋白进行反义下调是研究其正常功能的一种合适方法。
