Non-human animals having a genome comprising an engineered diversity cluster within an immunoglobulin heavy chain variable region and use thereof
An immunoglobulin and variable region technology, applied in immunoglobulins, peptides, chemical instruments and methods, etc.
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[0364] The following examples are provided to describe to those of ordinary skill in the art how to make and use the methods and compositions disclosed herein, and are not intended to limit the scope of what the inventors regard as their invention. Unless indicated otherwise, temperature is given in degrees Celsius and pressure is at or near atmospheric.
example 1
[0365] Example 1. Construction of engineered diversity clusters comprising nucleotide coding sequences for D6 chemokine decoy receptors within immunoglobulin heavy chain variable regions
[0366] This example illustrates an exemplary method for constructing a targeting vector for insertion into the genome of a non-human animal, such as a rodent (eg, mouse). In particular, the methods described in this example demonstrate the generation of targeting vectors for insertion into the genome of rodent (e.g., mouse) embryonic stem (ES) cells to produce immunoglobulin heavy chain variable The rodent of the immunoglobulin heavy chain variable region comprising engineered heavy chain diversity (D H ) area, the modified D H A region includes one or more nucleotide sequences each encoding a non-immunoglobulin polypeptide or a portion thereof. In this example, the coding sequence of the extracellular domain of an atypical chemokine receptor (ACKR), such as the D6 chemokine decoy receptor...
example 2
[0377] Example 2. Generation of Rodents with an Engineered Diversity Cluster Including the Nucleotide Encoding Sequence of a D6 Chemokine Decoy Receptor Within an Immunoglobulin Heavy Chain Variable Region
[0378] This example demonstrates the generation of a non-human animal (e.g., a rodent) whose genome comprises an immunoglobulin heavy chain variable region comprising an engineered heavy chain diversity (D H ) region, wherein the modified D H A region comprises one or more nucleotide sequences each encoding a portion of a non-immunoglobulin polypeptide, particularly an atypical chemokine receptor (ACKR), eg, the extracellular portion of the D6 chemokine decoy receptor.
[0379] The D6-D described in Example 1 was confirmed by sequencing using the primers set forth in Table 6 and by polymerase chain reaction throughout the construction of the targeting vector H Proper assembly of the targeting vector and targeted insertion of the D6 DNA fragment within the diversity cluste...
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