site-specific integration
A site, recombinant target site technology, applied in the direction of specific peptides, peptides, immunoglobulins, etc., can solve the problems of laborious, laborious screening mechanism, and lengthy construction of productive cell lines
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Embodiment 1
[0100] A) Materials and methods
[0101] 1. Vector construction
[0102] All vector sequences are synthesized and fully sequenced. Puromycin acetyltransferase (PAC), hygromycin phosphotransferase (Hyg) and mAb genes are fully optimized before gene synthesis and adapt to gray hamster codon preferences. pRY17( figure 1 Most of) are derived from pCB72.3-GA-HC-LC (Kalwy et al. (2006), Mol Biotechnol. 34. pages 151 to 156). The sequence of wild-type FRT (F) and the recombinant sequence of mutant F5FRT (F5) present in all vectors are from Schlake and Bode (Schlake and Bode, 1994, Biochemistry 33: 12746-12751). The source of the template sequence (before gene synthesis, with or without gene optimization) is as follows: the F recombination sequence in the vector pRY17 and the linker are fused in frame ( figure 1 ) Was taken from the vector pFRT / lacZeo (Invitrogen). In-frame fusion between the methionine start codon in pRY21 and the F recombination sequence ( image 3 ) Was obtained fr...
Embodiment 2
[0157] A) Materials and methods
[0158] 1. Southern Blot
[0159] 5-10 μg of genomic DNA isolated from the second and fourth generations of each clone and purified with the Blood & CellCulture DNA Maxi kit of QIAGEN (Qiagen) was digested with restriction enzymes at 37° C. for 15 h. The digested DNA was extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol mixture (pH 8.0, 1:1 v / v), and then, after precipitation with chloroform and ethanol alone, in 0.5×TBE (50×TBE: Lonza) or 1×TAE (40mM Tris, pH 7.7, 2.5mM EDTA) buffer in 0.7% (w / v) agarose gel electrophoresis. The gel was transferred to Hybond-N membrane (Amersham) using a vacuum manifold basically according to the manufacturer's instructions (Appligene, Pharmacia). The Hybond-N membrane was fixed with UV, and it was placed in 5×Denhardt's, 6×SSC (1×SSC: 0.15M sodium chloride, 15mM sodium citrate) prepared from 50× stock solution (Sigma) and 10 % (W / v) SDS in hybridization buffer or in separate Rapid-hyb bu...
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