Site-specific integration
A site and recombination target site technology, applied in the direction of specific peptides, peptides, immunoglobulins, etc., can solve the problems of laborious, laborious screening mechanisms, lengthy and other problems in the construction of productive cell lines
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Embodiment 1
[0100] A) Materials and methods
[0101] 1. Vector construction
[0102] All vector sequences were synthesized and fully sequenced. Puromycin acetyltransferase (PAC), hygromycin phosphotransferase (Hyg) and mAb genes were gene-wide optimized and adapted to the codon preference of gray hamsters prior to gene synthesis. pRY17( figure 1 ) was derived from pCB72.3-GA-HC-LC (Kalwy et al. (2006), Mol Biotechnol. 34. pages 151 to 156). The sequences of wild-type FRT (F) and mutant F5FRT (F5) recombinant sequences present in all vectors are from Schlake and Bode (Schlake and Bode, 1994, Biochemistry 33:12746-12751). Template sequences (before gene synthesis, with or without gene optimization) were sourced as follows: in-frame fusion of the F recombination sequence and linker in vector pRY17 ( figure 1 ) was obtained from the vector pFRT / lacZeo (Invitrogen). In-frame fusion of the methionine initiation codon and F recombination sequence in pRY21 ( image 3 ) was obtained from p...
Embodiment 2
[0157] A) Materials and methods
[0158] 1. Southern blot
[0159] 5-10 μg of genomic DNA isolated from passages 2 and 4 of each clone and purified with the Blood & Cell Culture DNA Maxi kit from QIAGEN (Qiagen) was digested with restriction enzymes for 15 h at 37°C. Digested DNA was extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol mixture (pH 8.0, 1:1 v / v), followed by separate chloroform and ethanol precipitations in 0.5×TBE (50×TBE: Lonza) or 1×TAE (40 mM Tris, pH 7.7, 2.5 mM EDTA) buffer on 0.7% (w / v) agarose gel electrophoresis. Gels were transferred onto Hybond-N membranes (Amersham) using a vacuum manifold essentially according to the manufacturer's instructions (Appligene, Pharmacia). Hybond-N membranes were UV-fixed and placed in 5×Denhardt’s, 6×SSC (1×SSC: 0.15M sodium chloride, 15 mM sodium citrate) and 10 % (w / v) SDS in hybridization buffer or prehybridized in Rapid-hyb buffer alone (GE healthcare).
[0160] Generate TK probes in PCR us...
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