Synthesis of a probe for monitoring HSV1-tk reporter gene expression using chemical exchange saturation transfer MRI

• Published in: Nature protocols Vol. 8; no. 12; pp. 2380 - 2391
• Main Authors: Bar-Shir, Amnon, Liu, Guanshu, Greenberg, Marc M, Bulte, Jeff W M, Gilad, Assaf A
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01.12.2013; Nature Publishing Group
• Subjects: 631/1647/1888; 631/1647/245/1628; 631/1647/666; Analytical Chemistry; Animals; Biological Techniques; Biosensors; Computational Biology/Bioinformatics; Data acquisition; Data processing; Enzymes; Gene expression; Genes, Reporter; Genetic research; Herpes simplex virus 1; Herpes viruses; Herpesvirus 1, Human - enzymology; Hydrogenation; Identification and classification; Kinases; Life Sciences; Magnetic Resonance Imaging - methods; Male; Mice; Microarrays; Monitoring methods; Organic Chemistry; protocol; Rodentia - virology; Rodents; Thymidine - analogs & derivatives; Thymidine - analysis; Thymidine - chemical synthesis; Thymidine - chemistry; Thymidine Kinase - genetics; Thymidine Kinase - metabolism; Tomography; Viral Proteins - analysis; United States > US; Maryland; Baltimore Maryland
• ISSN: 1754-2189; 1750-2799; 1750-2799
• DOI: 10.1038/nprot.2013.140

In experiments involving transgenic animals or animals treated with transgenic cells, it is important to have a method to monitor the expression of the relevant genes longitudinally and noninvasively. An MRI-based reporter gene enables monitoring of gene expression in the deep tissues of living subjects. This information can be co-registered with detailed high-resolution anatomical and functional information. We describe here the synthesis of the reporter probe, 5-methyl-5,6-dihydrothymidine (5-MDHT), which can be used for imaging of the herpes simplex virus type 1 thymidine kinase (HSV1 -tk ) reporter gene expression in rodents by MRI. The protocol also includes data acquisition and data processing routines customized for chemical exchange saturation transfer (CEST) contrast mechanisms. The dihydropyrimidine 5-MDHT is synthesized through a catalytic hydrogenation of the 5,6-double bond of thymidine to yield 5,6-dihydrothymidine, which is methylated on the C-5 position of the resulting saturated pyrimidine ring. The synthesis of 5-MDHT can be completed within 5 d, and the compound is stable for more than 1 year.