OBJECTIVE: To produce a chimaeric version of the 11-285-14 @entity787 ( @entity787 ) monoclonal antibody using a gene targeting approach. MATERIALS AND METHODS: A replacement vector was constructed to insert the @entity1 constant gamma1 gene within the @entity19 heavy chain locus of 11-285-14 hybridoma cells. The @entity19 constant gamma1 gene (1.5 kb) and the @entity19 mu intron fragment (2.2 kb) were amplified by PCR and cloned into a pKO Scrambler vector. The @entity1 constant gamma1 gene fragment (2.2 kb) was cloned next to the intron fragment. Resistant colonies were screened by ELISA for the presence of the @entity1 isotype in their supernatants. RESULTS: Of the 4,370 resistant colonies obtained, 87 colonies showed secretion of the @entity1 isotype at levels between 4 and 32 ng/ml. PCR and Southern blot results confirmed the correct integration of the @entity1 gene by homologous recombination within the heavy chain locus. Most of the producers ceased to express the @entity1 isotype within a few weeks after the initial positive ELISA results. Instability of secretion could not be explained by genetic instability in all the clones, which suggests the presence of other undefined epigenetic or physiologic mechanisms. CONCLUSION: Gene targeting resulted in transformants with unstable and low production rates of chimaeric anti- @entity787 antibody.

Instability of chimaeric antibody secretion by XXXX producing hybridoma cells after gene targeting.

@entity1 :: ('9606', 'Species') :: ['human'] @entity19 :: ('10090', 'Species') :: ['mouse'] @entity787 :: ('1084', 'Gene') :: ['CEA', 'anti-carcinoembryonic antigen']
@entity787 :: (1084,Gene) :: ['anti-carcinoembryonic antigen']
