Antibody Production,Regulation Diversity Southern Blotting to Detect lg Genes Hybrid Cell DNA Extraction Cleaved into fragments with restriction enzymes Separation by electrophoresis ■ Transferred Nitrocellulose ■■ 。 Incubate with P32labeled mRNA or cDNA probe (known, or H chain genes) onta e that hybridize with probe Immunoglobulin Genes Somatic cell hybridization techniques have been used to determine the chromosomal location of antibody genes.Murine B lymphocytes may be fused to another mammalian cell line to form unstable hybrids whose e chromosom s are randomly lost.The DNA of each hybrid is then probed ith radiolabeled copy DNA corresponding to kappa,lambda,or H chain regions for the presence of complementary H or L chain antibody genes.By correlating loss of a particular mouse chromosome with loss of a particular antibody gene,we can determine the chromosomal location of gene families. Using this approach,it became clear that three unlinked families of genes encode H and L chains and are located on separate chromosomes.Each family consists of a
Antibody Production, Regulation & Diversity Immunoglobulin Genes Somatic cell hybridization techniques have been used to determine the chromosomal location of antibody genes. Murine B lymphocytes may be fused to another mammalian cell line to form unstable hybrids, whose chromosomes are randomly lost. The DNA of each hybrid cell line is then probed with radiolabeled complementary, or copy, cDNA corresponding to kappa, lambda, or H chain regions for the presence of complementary H or L chain antibody genes. By correlating loss of a particular mouse chromosome with loss of a particular antibody gene, we can determine the chromosomal location of gene families. Using this approach, it became clear that three unlinked families of genes encode H and L chains and are located on separate chromosomes. Each family consists of a
cluster of gene segments capable of encoding V regions separated from one or more genes capable of encoding C regions. Chain Class Human Mouse Kappa 2 16 Lambda 22 6 H chain 14 12 Rearrangement of v and C Genes During Differentiation Mouse Embryo Cells Adult Mouse Mueloma cells ●● DNA Extracted. 2 .Restriction Endonucleases V 业业 11 听 Incubate with Ig mRNA ompryonic Hyeloma 6raiv/ Hybridization experiments revealed that V region and C region gene segments were separately encoded and located on distinct DNA fragments in embryonic cells,but on the same fragment in mature plasma cells.These studies suggested that DNA rearrangements must have occurred during maturation and differentiation of B lymphocytes.These rearranged DNA segments were destined to be expressed as specific monospecific eptors by antibody forming plasma cells.From the collection of gen segments available in each family,several may be specifically joined to encode an
cluster of gene segments capable of encoding V regions separated from one or more genes capable of encoding C regions. ______________________________________ Chain Class Human Mouse ______________________________________ Kappa 2 16 Lambda 22 6 H chain 14 12 ______________________________________ Hybridization experiments revealed that V region and C region gene segments were separately encoded and located on distinct DNA fragments in embryonic cells, but on the same fragment in mature plasma cells. These studies suggested that DNA rearrangements must have occurred during maturation and differentiation of B lymphocytes. These rearranged DNA segments were destined to be expressed as specific monospecific receptors by antibody forming plasma cells. From the collection of gene segments available in each family, several may be specifically joined to encode an
entire antibody chain L or H.It should be note that genes of one family were never intermingled with genes of either of the other families. Extracted germline DNA from mouse embryos and differentiated DNA from myeloma cells were cleaved with restriction enzymes.separated, denatured to yield single-stranded fragments,and extracted.Fragments were hybridized with kappa chain radiolabled probe (mRNA),derived from myeloma cells. The entire mRNA molecule (encoding the V and C regions)or only the 3'half of mRNA (encoding the C region)was employed.Results showed that mRNA for IgL chain hybridized to germline DNA by its Vregion to one DNA fragm to a totally separate DNA fragment.In contrast,the same mRNA hybridized to both V and C regions in a single DNA fragment from fully differentiated cells. Cloning of Specific Ig gene Mouse Myeloma Cell Bacteria (o ve DNA and restriction inte 85 and k With the advent of recombinant DNA technology,it became possible to clone specific antibody genes.Sufficient quantities of specific DNA segments could be obtained, permitting analysis of their organization.Studies of DNA sequence showed that discontinuous segments existed that did not code for peptides. Some noncoding segments were deleted from the DNA before transcription and others from the mRNA after transcription
entire antibody chain L or H. It should be note that genes of one family were never intermingled with genes of either of the other families. Extracted germline DNA from mouse embryos and differentiated DNA from myeloma cells were cleaved with restriction enzymes. Fragments were separated by electrophoresis, denatured to yield single-stranded fragments, and extracted. Fragments were hybridized with kappa chain radiolabled probe (mRNA), derived from myeloma cells. The entire mRNA molecule (encoding the V and C regions) or only the 3' half of mRNA (encoding the C region) was employed. Results showed that mRNA for Ig L chain hybridized to germline DNA by its V region to one DNA fragment and by its C region to a totally separate DNA fragment. In contrast, the same mRNA hybridized to both V and C regions in a single DNA fragment from fully differentiated cells. With the advent of recombinant DNA technology, it became possible to clone specific antibody genes. Sufficient quantities of specific DNA segments could be obtained, permitting analysis of their organization. Studies of DNA sequence showed that discontinuous segments existed that did not code for peptides. Some noncoding segments were deleted from the DNA before transcription and others from the mRNA after transcription
Tailor-Made Monoclonal Antibodies Mouse Ig genes Human Ig genes 隔WWT2cT CHI CH2 CH3 DJ CHI CH2 CH3 Splice and ligate 口 CH CHZ H .Mouse-human chimeric gene in vector Transfect with H chain Select for (penotes p of chimeric la gene) Production of human-mouse chimeric antibody The pioneering work of Kohler and Milstein in producing hybridomas has given rise to entirely new areas of immunodiagnosis and immunotherapeutics.It is now possible to produce monoclonal antibodies for use as probes and potential therapeutic agents. There are numerous technical problems that make production of therapeutic human monoclonals generally unsatisfactory.Mouse monoclonals are foreign proteins. Antibody genes form mice and humans can be isolated cloned and recombined.The tailor-made genes can then be inserted into suitable vector and transfected into meylomas.The resulting products have been called transfectomas.These chimeric monoclonals can be administered to humans
The pioneering work of Kohler and Milstein in producing hybridomas has given rise to entirely new areas of immunodiagnosis and immunotherapeutics. It is now possible to produce monoclonal antibodies for use as probes and potential therapeutic agents. There are numerous technical problems that make production of therapeutic human monoclonals generally unsatisfactory. Mouse monoclonals are foreign proteins. Antibody genes form mice and humans can be isolated, cloned and recombined. The tailor-made genes can then be inserted into suitable vector and transfected into meylomas. The resulting products have been called transfectomas. These chimeric monoclonals can be administered to humans
Recombination of Y-J gene segment of the Kappa chain 5 Germline DNA 阝 L YK2 L J1J2J3J4J5 Ck Rearrangement of DNA 5'B cell DNA LYKI L YK2 LYK3J2J3J4J5 Primary RNA Transcription 】R%下 VK3动2J34J5 A-A-A- VK3 2C Poly A tail Light Chain Gene Organization There are three peptide segments in each L chain and are coded by three distinct genes, V,J,and C.In the kappa family there are a large number of V gene segments and five joining (J)segments and a single C segn ment.By ontrast,in the lambda family,there are fewer V gene segments and a similar number of J genes but several C genes.The V and J gene segments encode V regions and C genes encode C regions.The L chain is encoded by three distinct gene segments that must be joined to form a functional antibody gene. Joining of Gene Segments During the r ess of differentiation of a pluripotent stem cell into a mature antigen reactive B cell,DNA rearrangements take place.In the kappa family one of the many B gene segments will become joined directly to one of the J gene segments.The V-J segment will be joined to the C segment to form a functional antibody gene.The joining of these segments is brought about through the activity of recombinase enzymes. Each germline V gene contains a region encoding the V segment and a leader sequence The leader sequence encodes a leader peptide that controls passage of new protein
Light Chain Gene Organization There are three peptide segments in each L chain and are coded by three distinct genes, V, J, and C. In the kappa family there are a large number of V gene segments and five joining (J) segments and a single C segment. By contrast, in the lambda family, there are fewer V gene segments and a similar number of J genes but several C genes. The V and J gene segments encode V regions and C genes encode C regions. The L chain is encoded by three distinct gene segments that must be joined to form a functional antibody gene. Joining of Gene Segments During the process of differentiation of a pluripotent stem cell into a mature antigen reactive B cell, DNA rearrangements take place. In the kappa family one of the many B gene segments will become joined directly to one of the J gene segments. The V-J segment will be joined to the C segment to form a functional antibody gene. The joining of these segments is brought about through the activity of recombinase enzymes. Each germline V gene contains a region encoding the V segment and a leader sequence. The leader sequence encodes a leader peptide that controls passage of new protein