All fractures are transformed into two part subtrochanteric fractures, and a DHS inserted using the standard technique with the plate fixed to the distal fragment using cortical screws.
For pM6P.blast iRhom2-3xHandnd pM6P.blast cub-3xHA, corresponding coding sequences were PCR amplified, the plasmid backbone was digested with restriction enzyme sites NcoI/XhoI and NcoI/NotI, respectively and PCR products were inserted using the InFusion cloning system (Clontech).
First, VL gene fragments for the κ (kappa) and λ (lambda) were inserted using the restriction sites MluI and NotI followed by cloning of the VH gene fragments via SfiI and HindIII.
For use, the osteochondral fragments are temporarily fixated using a K-wire and the Ethipin subsequently inserted using the application shell.
Human NRP1WT, HA-tagged GIPC1, V5-tagged Syx, HA-tagged constitutively active RhoA, and soluble NRP constructs were inserted using the pcDNA 3.1 TOPO expression vector (Life Technologies).
K15CreER(G)T2 was generated by inserting a 5-kb K15 promoter fragment (Liu et al, 2003) that was amplified and inserted using SalI restriction sites into the pK14CreER(G)T2 expression cassette after removal of the K14 regulatory sequence (Indra et al, 1999).
The CARD of Card9 (residues 6-100), tandemndem CARDs of NOD2 (residues 28-215) and the RIP2 CARD (residues 432-534) were amplified by PCR and inserted, using Gateway® cloning, into the expression plasmid pDEST-HisMBP [23].
After manual screening for additional contaminating sequences, 1632 cDNA clones were submitted for sequencing of the full length inserts using the forward and reverse primers of the pBluescript plasmid (T3, T7) to the Cincinnati Childrens Hospital Medical Center Sequencing Core, Cincinnati, OH.
The term hybrid fixation refers to implant fixation where one component is inserted using cement and the other without cement.
Target genes have to be inserted using restriction endonucleases and the plasmids do not encode cleavable affinity purification tags.
