Production of FMDV-resistant livestock by allele substitution
Inventors
CARLSON, DANIEL F. • Fahrenkrug, Scott C.
Assignees
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Publication Number
US-10058078-B2
Publication Date
2018-08-28
Expiration Date
Abstract
A genetically modified livestock animal comprising a genomic modification to an eIF4G gene. Cells, genes, and proteins encompassing a protease-resistant eIF4G protein or gene.
Core Innovation
The document describes genetically modified livestock, including a gene edited pig, whose genome comprises a modified eIF4GI gene having the nucleotide sequence of SEQ ID NO: 2. The modified eIF4GI gene expresses an altered eIF4GI protein relative to a wild type eIF4GI protein, and the altered eIF4GI protein is resistant to cleavage by a leader proteinase (Lpro) of foot-and-mouth disease virus (FMDV).
The background links FMDV infection to host translation shutoff by proteolytic cleavage of eukaryotic translation initiation factor 4G (eIF4G). The document characterizes protease-related cleavage by the FMDV leader proteinase (Lpro) and optionally also by 3C protease (3Cpro/Cpro), and describes protease-resistant eIF4G protein isoforms generated by minimal nucleotide edits in eIF4G/eIF4GI.
The document further emphasizes allele substitution, including transfer of natural alleles from other breeds/species, with precise site-specific changes while maintaining eIF4G function. It describes making the modified eIF4GI gene using site-specific nucleases such as TALENs, ZFNs, and CRISPR together with HDR templates, and it covers embodiments that use modified cells and animals for FMDV research and drug testing.
Claims Coverage
The document includes two independent claims: one product claim and one method claim. Across these claims, the inventive features focus on a modified eIF4GI nucleotide sequence (SEQ ID NO: 2) that generates an Lpro-resistant eIF4GI protein in a gene edited pig, and on producing that pig by TALEN-mediated editing with an HDR template followed by nuclear transfer into an oocyte and surrogate development.
Lpro-resistant modified eIF4GI in a gene edited pig
A gene edited pig whose genome comprises a modified eIF4GI gene having the nucleotide sequence of SEQ ID NO: 2, expressing an altered eIF4GI protein relative to a wild type eIF4GI protein such that the pig is resistant to cleavage by a leader proteinase (Lpro) of foot-and-mouth disease virus.
TALEN + HDR with SEQ ID NO: 2 and nuclear transfer to produce Lpro-resistant pigs
A method of producing a gene edited pig whose genome comprises a modified eIF4GI gene having the nucleotide sequence of SEQ ID NO: 2, wherein a TALEN pair specifically binds to the endogenous eIF4GI gene and causes a double-stranded DNA break, an HDR template comprising SEQ ID NO: 2 is introduced, homologous recombination events occur, the transfected fibroblast is transferred into an enucleated pig recipient oocyte for transgenic nuclear transfer embryo generation, the embryo is activated, and the embryo is transplanted into a surrogate mother pig.
Together, the independent claims cover a gene edited pig containing SEQ ID NO: 2-modified eIF4GI that yields an Lpro-resistant altered eIF4GI protein, and a production method using a TALEN pair with an HDR template comprising SEQ ID NO: 2, followed by transgenic nuclear transfer embryo generation and surrogate development to obtain the resistant pig.
Stated Advantages
The gene edited pig is resistant to cleavage by a leader proteinase (Lpro) of foot-and-mouth disease virus.
Documented Applications
Use of modified cells and animals for FMDV research and drug testing.
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