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Production of fmdv-resistant livestock by allele substitution

a technology of allele substitution and livestock, applied in the field of genetically modified animals, can solve the problems of poor fmdv resistance, lameness and appetite loss, pain in blisters, and mortality of young animals, and achieve the effects of reducing the number of dead animals, and improving the quality of li

Inactive Publication Date: 2018-11-01
RECOMBINETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention relates to cells, organisms, or animals that have a foreign gene that produces a protein called EIF4G. This protein helps fight a viral infection, called FMDV, by reducing its ability to bind to cells. By adding this foreign gene to animals, they can better protect themselves from FMDV. The invention also includes a method of modifying an organism's genetic material to include the foreign EIF4G gene. Overall, this technology helps create animals that are better equipped to resist viral infections.

Problems solved by technology

FMD infection of cattle and pigs causes fever, painful blistering, lameness and loss of appetite.
Mortality can occur in young animals due to infection of the heart muscle causing circulatory failure.
The disease is so highly contagious that infection in a single animal calls for the destruction and burial of the entire herd.
In 2001 an FMD outbreak in Great Britain resulted in total losses of about $12-4 billion [1] and more than a decade ago, the University of California Davis estimated that an FMD outbreak just in California could cost from $6-14 billion in control costs and lost markets due to restrictions in movement and sales of animals.
Sales of milk and other products, as well as meat, would be halted and jobs of producers and workers in related industries would be lost or severely curtailed.

Method used

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  • Production of fmdv-resistant livestock by allele substitution
  • Production of fmdv-resistant livestock by allele substitution

Examples

Experimental program
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example 1

[0130]Referring to FIG. 2, a portion of porcine EIF4GI is shown in panel A. The wild type sequence has asparagine and leucine residues in the minus 3 and 2 positions relative to the Lpro cleavage site (arrowhead). In this example, the HDR template replaces the minus 3 and 2 residues with aspartic acid and phenylalanine to render the modified EIF4GI resistant to Lpro cleavage. Two pairs of TALENs (top) were designed to cut the wild type EIF4GI to stimulate homologous recombination. Panel B: Surveyor (Cel-I) assay of pig fibroblasts transfected with each TALEN pair. Panel C: RFLP assay to determine the efficiency of homologous recombination.

[0131]Transfections were performed in early passage (<2 passages) primary pig fibroblasts. Fibroblasts at 70-90% confluence were harvested by for use in transfections. Two micrograms of TALEN mRNA ssEIF4G14.1 or ssEIF4G14.2 along with 0.2 nMole of a 90-mer homologous oligonucleotide (5′-cccagacttcactccgtectttgccgactteggccgaccagccatagcaaccgtgggccccc...

example 2

[0133]For colony isolation, cells will be enumerated and plated at a range of densities 1-20 cells / cm2 on 10 cm dishes. Cells will be cultured for 10-15 days until individual colonies of 3-4 mm in diameter are present. Individual colonies are aspirated with a p-200 pipettor under gentle aspiration and expelled into a well of 24-well plate with 500 μl of growth medium (Carlson, Garbe et al. 2011). Plates with clearly defined colonies (˜10-30 / plate) will be chosen for colony aspiration to limit the chance of aspirating cells from multiple colonies. Once a colony reaches 70-90 percent confluent in the 24-well dish, a portion will be harvested for RFPL analysis and the remainder will be cryopreserved. Cells will be taken from colonies that have been determined to have successfully acquired the intended features and used for cloning to make founder animals.

[0134]The specific embodiments above are intended to be illustrative and not limiting. Additional embodiments are within the broad co...

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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.

Description

CROSS REFERENCE TO RELATED APPLICATIONS[0001]This patent application is a continuation of U.S. application Ser. No. 13 / 836,860, filed Mar. 15, 2013 which claims priority to U.S. Provisional Application No. 61 / 677,904 filed Jul. 31, 2012, which is hereby incorporated by reference herein.SEQUENCE LISTING[0002]The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 11, 2018, is named 53545_710_301_SL.txt and is 4,179 bytes in size.TECHNICAL FIELD[0003]The technical field relates to genetically modified animals and associated techniques.BACKGROUND[0004]Cloven-hoofed animals infected by Foot and Mouth Disease Virus (FMDV) become rapidly incapacitated by acute vesicular disease. FMD infection of cattle and pigs causes fever, painful blistering, lameness and loss of appetite. As suggested by the name of the infectious agent, secondary infections of the ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A01K67/027C07K14/47
CPCA01K2267/02A01K2227/108A01K67/0275A01K2217/05C07K14/47A01K2227/101C12N15/8509
Inventor CARLSON, DANIEL F.FAHRENKRUG, SCOTT C.
Owner RECOMBINETICS INC
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