Technical Reference #1792

1792.

Designed Auto-assembly of Nanostreptabodies for Rapid Tissue-specific Targeting in Vivo Philippe Valadon; Bryan Darsow; Tim N. Buss; Malgorzata Czarny; Noelle M. Griffin; Han N. Nguyen; Phil Oh; Per Borgstrom; Adrian Chrastina; Jan E. Schnitzer, Proteogenomics Research Institute for Systems Medicine, Journal of Biological Cheimstry, 285(1792), (2010)
Link To Paper

Materials & Methods:
Vector Construction-tSK Vector Series—The Fc-encoding DNA of the human IGHG1 locus was cloned from HMVEC cells (Lonza) and after removal of the SfiI site located in the second intron by overlapping PCR transferred into the miniantibody vector mSK1 (33) to replace the equivalent murine Fc part. The resulting vector had the same mammalian/bacterial hybrid leader peptide with a double SfiI cloning site (5 -GGCCCAGCCGGCCATGCTAGTGGCCCGGGAGGCC) followed by the IGHG1 hinge region the CH2 domain and the CH3 domain together with introns. The construct was terminated by a SalI site and a His tag encoding the C-terminal sequence VDH6 in place of the CH3 terminal sequence PGK. The cassette was amplified by PCR with a primer adding a NotI site and BglII site after the stop codon and cloned after EcoRIBglII restriction into the EcoRI-BamHI fragment of the PTT3 episomal vector generously provided by Dr. Y. Durocher (47) to give the tSK-Fc vector. For the heavy chain vector the IGHV1.2 leader sequence (Fig. 1) was assembled from 4 overlapping primers with an EcoRI site on the 5 -side and an MfeI site followed by a NotI site on the 3 -side and transferred into the PTT3 vector as above. The human CH1 domain was amplified from genomic DNA assembled with the hinge-CH2-CH3 genomic fragment by overlapping PCR and cloned between the MfeI and the NotI sites to give the tSK-HC vector. The light chain vector was similarly constructed. The IGKV3–20 leader sequence (Fig. 1) was assembled from six overlapping primers with an EcoRI site on the 5 -side and an XbaI site and a NotI site on the 3 -side. After transfer into the PTT3 vector the CK domain was amplified from genomic DNA and cloned as an XbaI-NotI fragment to give the tSK-LC vector. Initial attempts using polyethylenimine transfection of 293-EBNA cells only yielded poor expression levels.

Microscopic Technique
Confocal Microscopy