Detection of H-2 alleles by PCR-RFLP-an alternative to flow cytometry in the screening of transgenic mice.
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.
Department of Visceral and Transplantation Surgery & Clinical Chemistry, Hannover University Medical School, 30625, Hannover, Germany.
Sven H G
SH
Klebs
Matthias W
MW
Hoffmann
Petra B
PB
Musholt
Bettina
B
Bayer
Thomas J
TJ
Musholt
D000483:Alleles; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D003429:Cross Reactions; D005434:Flow Cytometry; D006183:H-2 Antigens / Q000235:genetics* / Q000276:immunology; D020180:Heteroduplex Analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic / Q000235:genetics* / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D006183:H-2 Antigens; C024755:H-2K(K) antigen; C032876:H-2Kb protein, mouse