ALTERING CENTRAL-NERVOUS-SYSTEM PHYSIOLOGY WITH A DEFECTIVE HERPES-SIMPLEX VIRUS VECTOR EXPRESSING THE GLUCOSE TRANSPORTER GENE
ALTERING CENTRAL-NERVOUS-SYSTEM PHYSIOLOGY WITH A DEFECTIVE HERPES-SIMPLEX VIRUS VECTOR EXPRESSING THE GLUCOSE TRANSPORTER GENE
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DOI：
10.1073/pnas.90.8.3655
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发表时间：
1993-04-15
影响因子：
11.1
通讯作者：
SAPOLSKY, RM
中科院分区：
文献类型：
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作者：
HO, DY;MOCARSKI, ES;SAPOLSKY, RM
由于其有丝分裂后的性质，神经元是基因转移的困难对象。为了避免这种情况，我们使用了有缺陷的单纯疱疹病毒载体，在人巨细胞病毒 ie1 启动子的控制下过表达大鼠脑葡萄糖转运蛋白 (GT) 基因。该载体被命名为 vIE1GT，使用 1 型单纯疱疹病毒温度敏感突变体 ts756 进行繁殖。 vIE1GT 表达的 GT 很容易从 vIE1GT 感染的 Vero 细胞的膜组分中进行免疫沉淀。通过使用间接双重免疫荧光技术，vIE1GT 被证明能够增强培养的海马神经元和神经胶质细胞中 GT 的表达。相对于对照，此类 vIE1GT 感染培养物中的葡萄糖转运几乎增加了 2 倍。然后通过将 vIE1GT 显微注射到成年大鼠海马中来测试该系统的体内功效。 2 天后进行检查，通过原位杂交在海马神经元中证实了 vIE1GT 的 GT 表达；通过 2-脱氧[C-14]葡萄糖摄取和放射自显影，在注射部位周围的组织中检测到葡萄糖转运小幅但显着的增加。这种注射不会引起明显的细胞病理学变化。因此，这种方法可用于在体外和体内改变中枢神经系统生理学。
Because of their postmitotic nature, neurons are difficult subjects for gene transfer. To circumvent this, we have used a defective herpes simplex virus vector to overexpress the rat brain glucose transporter (GT) gene under the control of the human cytomegalovirus ie1 promoter. This vector, designated vIE1GT, was propagated using a herpes simplex virus type 1 temperature-sensitive mutant, ts756. GT expressed from vIE1GT was readily immunoprecipitated from membrane fractions of vIE1GT-infected Vero cells. By using indirect double immunofluorescence techniques, vIE1GT was shown to be capable of enhancing GT expression in cultured hippocampal neurons and glia. Glucose transport in such vIE1GT-infected cultures was increased almost-equal-to 2-fold relative to controls. The efficacy of this system in vivo was then tested by microinjection of vIE1GT into adult rat hippocampus. When examined 2 days later, GT expression from vIE1GT was demonstrated in hippocampal neurons by in situ hybridization; a small but significant increase in glucose transport was detected in tissue immediately surrounding the injection site by 2-deoxy[C-14]glucose uptake and autoradiography. Such injections did not cause marked cytopathology. Thus, this approach can be used to alter central nervous system physiology in vitro and in vivo.