方便且可重现的活体小鼠腮腺基因转移。
Convenient and reproducible in vivo gene transfer to mouse parotid glands.
机构信息
Molecular Physiology and Therapeutics Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-1190, USA.
出版信息
Oral Dis. 2011 Jan;17(1):77-82. doi: 10.1111/j.1601-0825.2010.01707.x.
OBJECTIVES
Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands.
METHODS
The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR.
RESULTS
The chosen volume for mouse parotid vector delivery was 20μL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva.
CONCLUSION
These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models.
目的
已发表的关于将基因转移到小鼠唾液腺的研究尚未采用腮腺。腮腺是大多数唾液腺基因转移临床应用的可能靶组织。本研究的目的是开发一种方便且可重复的向小鼠腮腺逆行基因转移方法。
方法
通过直接口腔插管将甲苯胺蓝注入 Stensen 导管后，评估载体输送的体积。构建了编码萤火虫荧光素酶或人红细胞生成素（hEpo）的重组 5 型腺病毒载体，然后将其施用于腮腺（10(7)载体颗粒/腺）。通过酶活性（荧光素酶）或酶联免疫吸附测定（hEpo）测量体内转基因表达。通过实时定量（Q）PCR 测量载体的组织分布。
结果
选择的用于小鼠腮腺载体输送的体积为 20μL。通过 QPCR 和荧光素酶测定，很少有载体检测到靶腺外。在腺体（荧光素酶，hEpo）和血清及唾液（hEpo）中均可轻易检测到转基因表达。大多数分泌的 hEpo 都在唾液中检测到。
结论
这些研究表明，小鼠腮腺可以方便且可重复地作为基因转移的靶标，对于许多小鼠疾病模型的临床前研究应该是有用的。
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