Immune Netw.  2002 Dec;2(4):233-241. 10.4110/in.2002.2.4.233.

Isolation of Mouse Ig Heavy and Light Chain Genomic DNA Clones, and Construction of Gene Knockout Vector for the Generation of Humanized Xenomouse

• Lee HK
• Cha SH

• KMID: 1974523
• DOI: http://doi.org/10.4110/in.2002.2.4.233

Abstract

BACKGROUND: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome.
METHODS
Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain lamda genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse.
RESULTS
Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress.
CONCLUSION
Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea.

Keyword

Hybridoma fusion; monoclonal antibody; human antibody; immunoglobulin gene; transgenesis; gene targeting

MeSH Terms

Animals
Antibodies
Antibodies, Monoclonal
Biotechnology
Brain
Clone Cells*
DNA*
Embryonic Stem Cells
Gene Knockout Techniques*
Gene Targeting
Gene Transfer Techniques
Genes, Immunoglobulin
Genetic Engineering
Genome
Genomic Library
Homologous Recombination
Human Body
Humans*
Hybridomas
Indicators and Reagents
Korea
Mass Screening
Mice*
Mice, Knockout
Mice, Transgenic
Polymerase Chain Reaction
Rodentia
Antibodies
Antibodies, Monoclonal
DNA
Indicators and Reagents