Transgenic animal with enhanced immune response and method for the preparation thereof

A genetically modified, non-transgenic technology, applied in the field of immunology, can solve the problem of failing to point out the advantages of immune response

Active Publication Date: 2009-12-02
AGRI BIOTECH CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

They also failed to point out the advantage of FcRn overexpression for enhancing the immune response

Method used

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  • Transgenic animal with enhanced immune response and method for the preparation thereof
  • Transgenic animal with enhanced immune response and method for the preparation thereof
  • Transgenic animal with enhanced immune response and method for the preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0173] Example 1 - Isolation and characterization of a BAC clone carrying the bFcRn alpha chain gene (bFCGRT)

[0174] Isolation of BAC clones carrying bFCGRT

[0175] The 90αBAC was isolated from a bovine BAC library obtained from an adult (2-year-old) male New Jersey cattle (acquired from the ResourceCenter / Primary Database of the German Genome Project (RZDP), Max Planck Institute of Molecular Genetics, Berlin, Germany; http: / / www.rzdp.de / ) preparation. bFCGRT positive BAC clones were identified by PCR screening with primers specific for bFcRn alpha chain mRNA, [206-425bp; GenBank AF139106) with BFclS: 5'-CAGTACCACTTCACCGCCGTGT-3' (SEQ.ID.NO.: 1) and BFclas : 5'-CTTGGAGCGCTTCGAGGAAGAG-3' (SEQ.ID.NO.: 2) amplification], next, bFCGRT DNA was sequenced with primers that can anneal to the exons of the gene (Kacskovics et al., 2000). To analyze the flanking region upstream of bFCGRT, the BAC

[0176] The DNA was digested with BamHI and the digested DNA was separated on an aga...

Embodiment 2

[0185]Example 2 - Generation and Genotyping of Transgenic Mice Carrying Bovine BAC 128E04

[0186] Preparation of a 102 kb genomic insert from BAC clone 128E04 for microinjection and generation of transgenic mice containing bovine BAC 128E04

[0187] BAC (clone 128E04) DNA was prepared for microinjection by slightly modifying the protocol published by Schedl et al. (Schedl et al., 1996). Purified BACs (Qiagen plasmid purification for very low copy number plasmids) were digested with Not I (Fermentas) to release the insert, which was separated on a precast pulsed field 1% agarose gel. Gel pieces containing the insert were electrophoresed into LMP (low melting point) gels which were digested with Gelase (Epicentre). A Microcon YM50 (Millipore) column was used to elute the insert from the agarose. The insert was washed in a buffer suitable for microinjection (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, pH 8.0, 100 mM NaCl, with or without 0.03 mM spermine / 0.03 mM spermine (SIGMA)). t...

Embodiment 3

[0203] Example 3 - Effect of bovine FCGRT (bFCGRT) gene copy number on FcRn expression

[0204] Determination of Transgene Copy Number Using Real-time Quantitative Polymerase Chain Reaction

[0205] Real-time PCR is a quantitative and precise method for determining transgene copy number and zygosity in Tg animals (Tesson et al., 2002). The copy number of the 128E04 BAC transgene was determined by absolute quantification of bFCGRT and the internal standard mouse β-actin gene, as follows.

[0206] The copy number of the 128E04 BAC transgene was determined by TaqMan method using ABI Prism7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Primers and probe oligonucleotide sequences were designed by using default parameters (primers and probes shown in Table 3). The traditional phenol / chloroform method was used for DNA extraction from tail samples of hemizygous animals, which required an additional chloroform extraction step.

[0207] Table 3. Primers and prob...

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Abstract

The present invention provides a method for producing a transgenic (Tg) non-human animal capable of developing an enhanced humoral immune response against an antigen as compared to a non-transgenic control animal of the same species, comprising introducing into said non-human animal a genetic construct providing for enhanced MHC class I-related neonatal Fc receptor (FcRn) activity. Also provided a Tg non-human animal comprising a genetic construct providing for enhanced FcRn activity, as well as the use of such animal in a non- therapeutical method. Therapeutic genetic constructs and methods are also provided. The present invention further provides methods for producing immunoglobulins.

Description

technical field [0001] The present invention relates to the field of immunology. More specifically, the present invention provides a method for producing non-human transgenic (Tg) animals comprising introducing into said non-human In a human animal, the non-human transgenic animal is able to mount an enhanced humoral immune response to the antigen compared to a non-transgenic control animal of the same species. The invention also provides a non-human Tg animal comprising a genetic construct providing enhanced FcRn activity and the use of such an animal in non-therapeutic methods. Therapeutic genetic constructs and methods are also provided. The invention further provides methods for producing immunoglobulins. Background technique [0002] In a specific humoral immune response, plasma cells develop from B lymphocytes in response to antigen, which peaks approximately 1-2 weeks after antigen stimulation. The second encounter with the antigen results in secretion of antibodi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/705C12N15/85A01K67/027
CPCA01K67/0275A01K2217/05A01K2227/105A01K2227/107A01K2267/01A01K2267/03A61K39/00A61K48/00A61K2039/545A61K2039/55A61P37/00A61P37/04C07K14/70535C12N15/8509
Inventor 茹然瑙·伯塞伊姆雷·考奇科维奇尤迪特·切尔韦纳克拉斯洛·希里皮鲍拉日·本德尔
Owner AGRI BIOTECH CENT
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