By Wolfgang Walther, Ulrike Stein
This quantity offers perception into contemporary advancements on experimental and medical concepts for melanoma gene remedy. Gene remedy of sturdy Cancers: tools and Protocols courses readers throughprotocols on gene healing concepts together with valuable technical notes. Written within the hugely winning Methods in Molecular Biology series structure, chapters contain introductions to their respective issues, lists of the required fabrics and reagents, step by step, without difficulty reproducible laboratory protocols, and key tips about troubleshooting and fending off identified pitfalls.
Concise and easy-to-use, Gene treatment of stable Cancers: equipment and Protocols goals to make sure winning leads to the additional learn of this very important field.
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Additional info for Gene Therapy of Solid Cancers: Methods and Protocols
Sterile cuvettes with aluminum electrodes. Distance of electrodes of 4 mm is recommended. 3. Dishes (6 cm diameter) with low attachment surface: These dishes prevent cell adhesion and minimize protein absorption, enzyme activation, and cellular activation. 3 Methods Cell Cultivation Cultivate RCC cells under standard conditions at 37 °C, 5 % CO2, 98 % humidity in an incubator and split twice weekly in a 1:3 or 1:4 ratio. Use trypsin solution for detachment of cells from the cultivation flasks (see Note 6).
They are generally classified into viral and non-viral vectors. Recently, the non-viral MIDGE® (minimalistic immunogenically defined gene expression) vectors were designed and developed for clinical use [1–6]. MIDGE vectors are linear DNA molecules of covalently closed topology (Fig. 1). They are biotechnologically manufactured from plasmids, which were especially designed to allow for excision of the entire expression cassette and subsequent ligation of short hairpin oligodeoxynucleotides (ODN) in a one-vessel reaction.
Add 100 μl of ConA working solution into the positive control wells (3 wells). 12. Culture cells at 37 °C overnight in a 5 % CO2 incubator. 13. At day 3 detect the IFNg/IL-4 spots according to the manufacturer’s instructions. 14. Prepare DAB substrate solution during the last incubation step with streptavidin-HRP, and keep the solution on ice in the dark. 15. Add 50 μl DAB solution to each well, and incubate for up to 10 min in the dark to detect the spots (see Note 12). 16. Stop the reaction by washing with tap water.