Use of a simple, general targeting vector for replacing the DNA of the heavy chain constant region in mouse hybridoma cells.
It is often necessary to modify the constant region of the immunoglobulin (Ig) heavy chain in order to produce Ig with optimal properties. In the case of Ig production by mouse hybridoma cells, it is possible to modify the Ig heavy chain (IgH) locus by gene targeting to achieve the desired changes. DNA segments from the JH-S micro region and from the region 3' of Calpha are normally present in the functional IgH gene of all hybridomas, regardless of the heavy chain class which is expressed. Consequently, these DNA segments could in principle serve as 5' and 3' homology regions to create a "universal" targeting vector for replacing the constant region exons in the IgH locus of any hybridoma cell. The practicality of this vector design has been uncertain. That is, the extent of the chromosomal DNA which would be replaced by a universal targeting vector would be as little as 5 kb (in a cell producing the alpha heavy chain) and as much as 180 kb (in a micro -producing cell), and it has been uncertain whether it would be practical to generate such long chromosomal deletions by gene targeting. Using a vector of this design, we found (a) that correctly targeted recombinant cells lacking the 180 kb DNA segment occurred at a low but usable frequency, (b) that these recombinants expressed the modified IgH locus at the same rate as the original hybridoma and (c) that IgH expression in these cell lines was stable. Our results thus indicate that this vector design is suitable for modifying IgH loci expressing any heavy chain, provided that an efficient selection or screening for targeted recombinants is available.
Ronai
Diana
D
Immunology Department, University of Toronto, Toronto, ON, Canada M5S 1A8.
Cheng
Emily Y
EY
Collins
Catherine
C
Shulman
Marc J
MJ
D000818:Animals; D004274:DNA, Recombinant / Q000235:genetics; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain; D018390:Gene Targeting / Q000379:methods*; D005803:Genes, Immunoglobulin; D005822:Genetic Vectors; D006825:Hybridomas / Q000276:immunology; D007127:Immunoglobulin Constant Regions / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007148:Immunoglobulin mu-Chains / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D004274:DNA, Recombinant; D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D007148:Immunoglobulin mu-Chains; D011994:Recombinant Proteins