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Transgenic animal expressing hla-a24 and utilization thereof

Inactive Publication Date: 2005-03-03
SUMITOMO DAINIPPON PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0091] The purified HLA-A24 gene is then introduced into a fertilized egg of the subject animal. The subject animal may be specifically, for example, mouse, rat, rabbit, etc.; however, rodents such as mouse, rat, etc. are preferred from the viewpoint of feasibility of generation, fosterage and application, and mouse is especially preferred. Above all, a mouse of strain C57BL / 6 is more preferred. C57BL / 6 mouse strain has an advantage because said mouse expresses H-2K.sup.b as the class I molecule and not H-2K.sup.d having similar binding motif as HLA-A24. That is, when an HLA-A24-binding antigen is administered, there is no danger of presentation of an antigen on the cell surface or cross-reaction.
[0155] By returning the above therapeutic agent into the patient's body, the toxicity of CTLs on tumor cells is enhanced in the patient who is positive for both HLA-A24 and PSA efficiently and destroy the tumor cells, and thereby achieving the treatment of tumor. The CTLs of the present invention are especially useful in the treatment of prostatic cancer.

Problems solved by technology

Furthermore, it was totally unpredictable whether or not a genomic DNA of chimera HLA-A24 containing such an artificial sequence is spliced normally to render the expression of the intended chimera HLA-A24 molecule, when introduced into an individual.

Method used

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  • Transgenic animal expressing hla-a24 and utilization thereof
  • Transgenic animal expressing hla-a24 and utilization thereof
  • Transgenic animal expressing hla-a24 and utilization thereof

Examples

Experimental program
Comparison scheme
Effect test

example 2

Cloning of H-2K.sup.b Genomic DNA Fragment

[0169] (1) Cloning of H-2K.sup.b Genomic DNA Fragment

[0170] Mouse tumor cell line EL4 (ATCC T1B-39) was cultured, and mouse genomic DNA was purified and used in the PCR cloning. Purification of DNA was carried out using TaKaRa LA Taq.TM. (Takara Shuzo) suited for the amplification of long-chain DNA as per the attached protocol. The GenBank database was then searched for H-2K.sup.b gene needed for the construction of chimeric HLA gene, which revealed that said gene was divided in two segments registered under the Accession Nos. v00746 and v00747. As v00746, the upstream 1594 bp region of H-2K.sup.b down to midstream of intron 3 was registered and, as v00747, the downstream 1837 bp region of H-2K.sup.b down to midstream of intron 7 was registered. Because there was no BamHI restriction site in intron 3, which is divided and registered as v00746 and v00747, the H-2K.sup.b gene registered at the database was thought to have incomplete length.

[01...

example 3

Construction of Chimera Genomic DNA (HLA-A2402 / K.sup.b DNA)

[0187] The Plasmid HLA-A2402#1 containing HLA-A2402 genomic DNA obtained in Example 1 was cleaved at BglII restriction site and the plasmid H-2K.sup.b#20 / 26 containing H-2K.sup.b genomic DNA obtained in Example 2 was cleaved at BamHI restriction site, and the resultant fragments were ligated to give a recombinant plasmid. The schematic construction is shown in FIG. 2. The recombinant plasmid was introduced into E. coli JM109 (Toyobo) by heat shock method at 42.degree. C., and white colonies of E. coli to which the recombinant plasmid has been introduced were selected on ampicillin-containing LB agar medium coated with X-Gal and IPTG to obtain the transformants. Ten transformants were incubated overnight in LB medium containing ampicillin (3 ml). The plasmid clone contained in each transformant was purified and subjected to sequence analysis in a similar manner to the above. As a result, it was revealed that three transforman...

example 4

Splicing Analysis of Chimera Genomic DNA

[0188] Mouse tumor cell line EL4 was transfected with the constructed chimeric HLA gene (HLA-A2402 / K.sup.b gene) with Electro Gene Transfer GTE-10 (Shimadzu) as per the attached protocol. Two days later, total RNA was purified from transfected EL4 cells and un-transfected EL4 cells (control) by using ISOGEN (Nippon Gene) as per the attached protocol. Reverse transcription was performed using SuperScript Choice System (GIBCO BRL) as per the attached protocol using Oligo(dT).sub.12-18 and a part of said RNA as a template to synthesize cDNA. In addition, chimera gene was specifically amplified by PCR using Native Pfu DNA Polymerase (Stratagene) and a part of said cDNA as a template.

[0189] PCR was conducted using an upstream primer Chimera-F2:

[0190] 5'-CGA ACC CTC GTC CTG CTA CTC TC-3' (23 mer, SEQ ID NO:10), which is encoded in exon 1 of HLA-A2402 gene and has low homology with H-2K.sup.b gene, and a downstream primer Chimera-R2:

[0191] 5'-AGC ATA...

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Abstract

The present invention relates to a non-human transgenic mammal which has had an HLA-A24 gene introduced and in which CTLs are induced when stimulated by an HLA-A24-binding antigen, a method of screening therapeutic or preventive agents for tumors or virus infections comprising administering a test substance to said transgenic non-human mammal and assaying and evaluating whether CTLs specific for the test substance are induced, an HLA-A24-binding tumor antigen peptide of PSA origin selected by said screening method, a chimera DNA (DNA construct) useful in the generation of said non-human transgenic mammal, a host cell transformed by said chimera gene and use thereof, and the like.

Description

[0001] The present invention relates to transgenic animals expressing HLA-A24 and utilization thereof. More specifically, the present invention relates to transgenic animals, into which an HLA-A24 gene has been introduced and in which cytotoxic T lymphocytes are induced when stimulated by an HLA-A24-binding antigen (i.e., HLA-A24-restricted antigen), a method of screening therapeutic or preventive agents for tumors or virus infections using said transgenic animals, HLA-A24-binding tumor antigen peptides of PSA origin selected by said method, recombinant DNA constructs useful in the preparation of transgenic mice and the use thereof, and the like.BACKGROUD ART[0002] The cellular immunity, particularly cytotoxic T lymphocyte (hereinafter, referred to as "CTL"), plays an important role in the removal of cancer cells or virus-affected cells from living body. CTLs recognize a complex between a peptide fragment of antigen proteins originated from cancer, virus etc. and an MHC class I mole...

Claims

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Application Information

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IPC IPC(8): A01K67/027A61P31/12A61P35/00A61P43/00C07K14/74C12N15/85
CPCA01K67/0275A01K2217/05A01K2227/105C12N15/8509A01K2267/0393C07K14/70539C07K2319/00A01K2267/03A61P31/12A61P35/00A61P43/00
Inventor GOTOH, MASASHI
Owner SUMITOMO DAINIPPON PHARMA CO LTD
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