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Immunocompromised Ungulates

a technology of immunocompromised ungulates and ungulates, which is applied in the field of immunocompromised ungulates, can solve problems such as technical limitations, and achieve the effect of effective modeling human infectious disease pathologies

Inactive Publication Date: 2012-04-12
AYARES DAVID L +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0014]There remains a need for a true large animal model that lacks a humoral immune response and that can effectively model human infectious disease pathologies. The pr...

Problems solved by technology

Although mice are used extensively, there are few examples in the literature for the use of other species as immuno-deficient animal models for human disease research.
Although such mouse models have proven useful, researchers still stress that there are technical limitations, as animals which normally develop listeriosis are not classical laboratory animals such as the rat or mouse, but rather farm animals (Lecuit, (2007) Microbes Infect 9, 1216-1225).

Method used

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Examples

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example 1

Construction and Design of Hc KO Vector

[0167]A portion of the genomic porcine Ig Hc locus was cloned and characterized. FIG. 1 illustrates the architecture of the porcine Hc locus. The single functional JH was found to reside within 6 kb upstream of the coding sequence for the Mu constant (Cμ region). The porcine JH was identified as an ‘Achilles heel’ that could be deleted to produce only non-functional VDJ rearrangement and, thus, prevent B-cell survival.

[0168]A poly (A) trap targeting vector was constructed (pPL708, FIG. 1) by flanking a CMV enhancer-pgk-neoR cassette that lacked polyA signal sequences, with a 2 kb upstream arm and a 5.8 kb downstream arm of homology. The downstream arm retained the complete JH to Cμ intron, and a few by of 3′ JH sequence, to insure normal splicing between the neoR transcript and the poly(A) signals found downstream of the Cμ coding region.

Vector Construction. Hc sequence data required for construction of a porcine Hc gene-targeting vector was ge...

example 2

Heterozygous Hc KO in Porcine Cells

[0169]Isolation and transfection of primary porcine fetal fibroblasts. PCFF4-1 to PCFF4-10 PPFF cells were isolated from 10 fetuses of the same pregnancy at day 33 of gestation. After removing the head and viscera, fetuses were washed with Hanks' Balanced Salt Solution (HBSS, Invitrogen), placed in 20 ml of HBSS and diced with small surgical scissors. The tissue pellet was resuspended in 50 ml tubes with 40 ml of DMEM+100 U / ml collagenase (Invitrogen) per fetus. Tubes were incubated for 40 min in a shaking water bath at 37° C. The digested tissue was allowed to settle for 3-4 min and the cell-rich supernatant was transferred to a new 50 ml tube and spun down. The cells were then resuspended in 40 ml of DMEM, 10% Fetal Calf Serum, 1× non-essential amino acids, 1 mM sodium pyruvate (Invitrogen) and 2 ng / ml basic fibroblast growth factor (Roche Molecular Biochemicals, Indianapolis, Ind.), then seeded into 10 cm dishes. All cells were cryopreserved upo...

example 3

Somatic Cell Nuclear Transfer to Produce Heterozygous Hc KO Pigs

[0171]Somatic Cell Nuclear Transfer (SCNT) procedure. Cells from one of the three correctly targeted colonies obtained from transfection of pPL708 into female cell line PCFF4-8 were used as nuclear donors for SCNT. SCNT procedures were performed on in vitro matured oocytes (BoMed, Madison, Wis. and / or TransOVA, IA) using techniques described in the literature (Polejaeva, et al., (2000) Nature 407, 86-90, Dai et al., (2002) Nature biotechnology 20, 251-255, Campbell et al., (2007) Theriogenology 68 Suppl 1, S214-231, Vatja et al., (2007) Reprod Fertil Dev 19, 403-423). Electrical fusion and activation of reconstructed oocytes was performed using an ECM2001 Electrocell Manipulator (BTX Inc., San Diego). Fused nuclear transfer embryos were cultured in NCSU-23 medium for 1-4 h at 38.5° C., and then transferred to the oviduct of an estrus-synchronized recipient gilt. Crossbred gilts (large white / Duroc / Landrace) (280-400 lbs)...

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Abstract

Porcine animals, tissue and organs as well as cells and cell lines derived from such animals are provided that lack functional endogenous immunoglobulin loci and are deficient in immunoglobulin expression and B-cells. These animals are useful as model systems for research and for development of new pharmaceutical and biological agents. In addition, methods are provided to prepare such animals.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Provisional Application No. 61 / 108,742, filed Oct. 27, 2008, the disclosure of which is incorporated herein in its entirety.FIELD OF THE INVENTION[0002]Animals, in particular porcine animals, tissue and organs as well as cells and cell lines derived from such animals are provided that lack functional endogenous immunoglobulin expression. These animals are useful for development of new pharmaceutical and biological agents. In addition, methods are provided to prepare such animals.BACKGROUND[0003]Models are necessary to study certain disease characteristics. In infectious disease research and vaccine development, it is important to study the cellular and humoral immune response, both together and separately. A 2006 report by the World Health Organization stated “There is evidence suggesting that both cellular and humoral immune responses contribute to protection in humans, and thus the development of...

Claims

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Application Information

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IPC IPC(8): G01N33/483C12Q1/68C12Q1/02G01N33/566A01K67/027C12P21/00
CPCA01K67/0273A01K67/0275A01K67/0276A01K2217/052A01K2217/075G01N33/5088A01K2267/01A01K2267/0387C07K14/70521C07K2319/30C12N15/8509A01K2227/108C12N2015/8518C12N2015/8527
Inventor AYARES, DAVID L.MENDICINO, MICHAELWELLS, KEVINDANDRO, AMY
Owner AYARES DAVID L
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