Non-human animals with modified immunoglobulin heavy chain sequences
An immunoglobulin, human technology, applied in the direction of immunoglobulin, anti-animal/human immunoglobulin, microorganisms, etc.
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Embodiment 1
[0537] Example 1. Cloning and expression analysis of candidate universal heavy chain sequences
[0538] Previous studies have shown that hV H 3-23 is a thermostable human variable heavy chain gene segment and is also one of the most commonly used variable segments in the human lineage. Therefore, the codon-optimized human V H 3-23, D4-4 (reading frame 2 or 3), and J H 4 (or J H 6) Gene segments are used to design rearranged heavy chain variable sequences (hereinafter "universal heavy chain" or "UHC").
[0539] Briefly, the following four candidate rearranged VDJ sequences were synthesized de novo (by IDT) and cloned into CMV expression vectors (eg, pRG1301 or pRG1368): (1) hV H 3-23(D4-4_RF2)J H 4 (SEQ ID NO: 148); (2) hV H 3-23(D4-4_RF2)J H 6 (SEQ ID NO: 146); (3) hV H 3-23(D4-4_RF3)JH 4 (SEQ ID NO: 147); (4) hV H 3-23(D4-4_RF3)J H 6 (SEQ ID NO: 145). All of these constructs were designed in such a way that the synthetic UHC gene could be ligated into the pRG1301 (...
Embodiment 2
[0543] Example 2. Construction of Immunoglobulin Heavy Chain Loci Containing Rearranged VDJ Sequences
[0544] Immunoglobulin heavy chain loci containing rearranged human VDJ sequences were constructed by a series of homologous recombination reactions using bacterial artificial chromosome (BAC) DNA in bacterial cells (BHR). use Genetic engineering transformation technology (see for example U.S. Patent No. 6,586,251 and Valenzuela, D.M. et al. (2003), High-throughput engineering of the mouse genome coupled with high-resolution expression analysis (High-throughput engineering of the mouse genome coupled with high-resolution expression analysis), Nature Biotechnology 21 (6) :652-659, incorporated herein by reference in its entirety) to generate several targeting constructs for the formation of genetically engineered mice expressing rearranged heavy chain variable domains.
[0545] Briefly, targeting vectors were designed to incorporate rearranged human immunoglobulin heavy chai...
Embodiment 3
[0555] Example 3. Characterization of Genetically Modified Mice Expressing Rearranged Heavy Chain Variable Domains
[0556] All mice were housed and bred at Regeneron Pharmaceuticals under specified pathogen-free conditions. Three wild-type (WT) littermate control mice (16 weeks old, male, n=2; background: 75% C57 / BL6 and 25% 129) and two to four MAID6032HETF0 mice ( Figure 9 ; 9 weeks old, male, n=2; background: 50% C57 / BL6 and 50% 129) were sacrificed and blood, spleen and bone marrow were collected from the animals. Additionally, four wild-type (WT) littermate control mice (10 weeks old; 2 males and 2 females) and four MAID6032 homozygous ("HO") F2 mice (10 weeks old; 3 1 male; 1 female) were sacrificed and blood, spleen and bone marrow were collected from the animals. Blood was collected into BD microtainertubes with EDTA (Catalog #365973). Bone marrow was collected from femurs by flushing with complete RPMI medium (RPMI medium supplemented with fetal calf serum, sodiu...
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