Detection of H-2 alleles by PCR-RFLP - an alternative to flow cytometry in the screening of transgenic mice

被引:1
作者
Klebs, SHG [1 ]
Hoffmann, MW [1 ]
Musholt, PB [1 ]
Bayer, B [1 ]
Musholt, TJ [1 ]
机构
[1] Univ Hannover, Dept Visceral & Transplantat Surg & Clin Chem, D-30625 Hannover, Germany
关键词
histocompatibility antigens; MHC; RFLP; transgenic mice; H-2; antigens;
D O I
10.1016/S0022-1759(03)00063-2
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
An increasing number of experimental models is based on well-defined transgenic mice in medical and biological research. Particularly in settings in which transgenic recombinants are used, a fast and reliable method is needed to screen for a defined H-2 background. For this purpose, flow cytometry with specific monoclonal antibodies is the standard procedure. However, epitopes of closely related rodent strains show only minor variations affecting the production of specific discriminating antibodies. Therefore, cross-reactivity of antibodies against specific major histocompatibility complex (MHC) leads to unreliable results in settings with closely related strains. In need of a method with high reliability, we have designed a screening assay based on polymerase chain reaction (PCR) followed by restriction fragment length polymorphisms (RFLP) to discriminate the MHC class I antigens H-2K(d), -K-b, K-k, which are sequence variants of the H-2K gene. A part of the mus musculus MHC gene coding for H-2K-covering exons 4 and 5 with MHC-differentiating restriction sites-was amplified. Subsequent restriction digest of the PCR products allows to discriminate the three aforementioned alleles and to identify homozygous as well as heterozygous haplotypes. To distinguish transgenic mice defined by certain MHC backgrounds, the PCR-RFLP method is simple, cost-effective, specific, and reliable and can be used independently or in addition to other methods in any laboratory. (C) 2003 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:197 / 205
页数:9
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