Biochemical and Biophysical Research Communications
Positive and negative cooperativity of modularly assembled zinc fingers
Section snippets
Materials and methods
Chemicals. The modification enzymes and restriction enzymes were purchased from New England Biolabs, except for AgeI that was obtained from Nippon Gene. The Taq DNA polymerase was acquired from Nippon Gene. The synthesized oligonucleotides were supplied from Invitrogen. All other chemicals were of commercial reagent grade.
Construction of expression vectors and reporter vectors. The multi-cloning sites containing the XmaI, EcoRI, and BamHI restriction sites were introduced between the SacI and
Creation of 6-zinc finger proteins targeting a specific E-box
As the targets of the artificial zinc finger proteins, we selected the E-box-containing sequences on the mouse Period1 (mPer1) promoter involved in the regulation of the circadian clock. There are at least five E-box sequences (E1–E5) on the mPer1 promoter [19], but the contribution of each E-box is unclear. It is required to create site-specific zinc finger proteins that can recognize a specific E-box for future genomic promoter analyses. In order to target a specific E-box, we paid attention
Acknowledgments
This work was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan and Takeda Science Foundation to M.I. A.N. and T.M. are research fellows of the Japan Society for the Promotion of Science.
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