Ungulates with genetically modified immune systems
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Publication Number
US-11085054-B2
Publication Date
2021-08-10
Expiration Date
Abstract
The present invention provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which lack expression of functional endogenous immunoglobulin loci. The present invention also provides ungulate animals, tissue and organs as well as cells and cell lines derived from such animals, tissue and organs, which express xenogenous, such as human, immunoglobulin loci. The present invention further provides ungulate, such as porcine genomic DNA sequence of porcine heavy and light chain immunogobulins. Such animals, tissues, organs and cells can be used in research and medical therapy. In addition, methods are provided to prepare such animals, organs, tissues, and cells.
Core Innovation
The invention relates to ungulate animals and derived cells in which endogenous immunoglobulin loci are lacking functional activity. It provides ungulate immunoglobulin germline gene sequence arrangements and novel genomic sequences associated with porcine heavy chain, porcine kappa light chain, and porcine lambda light chain, including flanking and joining/constant region elements, to support locus characterization beyond known cDNA sequences.
The invention provides targeting vector concepts for gene targeting by homologous recombination. Targeting constructs are described for disrupting porcine immunoglobulin genetic loci, including porcine lambda light chain constructs and constructs directed to kappa constant regions and lambda J/C cluster regions, with flanking sequences, selectable markers, and recombination arms suitable for locus inactivation.
The invention further provides large artificial chromosomes, including megabase-scale AC constructs, engineered to carry human immunoglobulin loci such as human kappa light-chain locus and human heavy-chain locus. The ACs are introduced into ungulate cells, including porcine cells, and genetically modified animals are generated using the described constructs for nuclear transfer-based generation, with animals described as lacking functional immunoglobulins and/or expressing xenogeneic human Ig genes.
Claims Coverage
The consolidated claim coverage includes one independent claim directed to a targeting vector that inactivates a porcine lambda light chain genetic locus using 5′ and 3′ flanking nucleotide sequences around the J/C regions plus a selectable marker gene. Dependent claims refine the 3′ flank by position, minimum contiguous-nucleotide length, and SEQ ID NO-defined or homology-based sequence selection.
Porcine lambda light chain locus inactivation targeting vector with 5′ and 3′ J/C flanks
A targeting vector that inactivates a porcine lambda light chain genetic locus comprising a first nucleotide sequence with at least 30 contiguous nucleic acids flanking the 5′ end of the first joining/constant (J/C) region, a selectable marker gene, and a second nucleotide sequence with at least 30 contiguous nucleic acids flanking the 3′ end of the J/C cluster region.
Porcine lambda light chain 3′ flank positional constraint
The nucleotide sequence flanking the 3′ end of the J/C cluster region is approximately 200 base pairs downstream of the J/C region of a porcine lambda light chain genetic locus.
Expanded minimum contiguous nucleic acid length for 3′ flank
The first nucleotide sequence contains at least 1000 contiguous nucleic acids corresponding to the region flanking the 3′ end of the J/C cluster region.
Porcine lambda light chain locus defined by SEQ ID NO: 28
The targeting vector includes a porcine lambda light chain genetic locus defined by the nucleotide sequence SEQ ID NO: 28.
3′ flank 85% homology to SEQ ID NOs 33–39
The targeting vector includes a nucleotide sequence flanking the 3′ end of the J/C cluster region that is at least 85% homologous to the nucleotide sequence of any one of SEQ ID NOs 33–39.
3′ flank defined as SEQ ID NO: 33
The targeting vector includes, as the nucleotide sequence flanking the 3′ end of the J/C cluster region, the nucleotide sequence of SEQ ID NO: 33.
The claim coverage centers on a targeting vector for inactivating a porcine lambda light chain genetic locus, with inventive emphasis on 5′ and 3′ J/C region flanking sequences and a selectable marker gene, together with dependent refinements that impose positional, contiguous-length, and sequence-identity constraints on the 3′ flanking sequence.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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