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Standard molecular techniques used to make constructs were as described in Sambrook and Russell [ 48].
This feature also makes it extremely easy to make constructs for fusion proteins and chimeras.
The PCR products were then cloned into pcDNA3.1 (Invitrogen) using KpnI and XbaI restriction sites to make constructs pcDNA/Iso1 and pcDNA/Iso2.
Full-length K16 cDNA was cloned into expression vector pCR3.1 (Life Technologies) and used as a template for site-directed mutagenesis (Stratagene, Cedar Creek, TX, U.S.A). to make constructs containing K16 p.and418Cys and K16 p.Arg418Pro.
We then proceeded to make constructs containing this repressor domain (RD) and parts of this in frame with Gal4 DBD and used the constructs to assess the minimal RD of Gli3.
To make constructs resistant against PICH-1 siRNA (Baumann et al. 2007), the above constructs were then subjected to site-directed mutagenesis using the following primers: M3344-GAGGGTGAGAAACAAGACTTATCCAGTATAAAGGTG and M3345-CACCTTTATACTGGATAAGTCTTGTTTCTCACCCTC.
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FAP was inserted between NheI and BamHI sites to make construct FAPdL5:**.
Tunnels that differ from this design offer problems that make constructing them more complex.
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