DETERMINATION OF CELL ORIGIN AFTER MARROW TRANSPLANTATION IN CANINES BY POLYMERASE-CHAIN-REACTION AND QUANTITATION OF THE ZFY ZFX GENES/

Background. In order to follow the course of bone marrow engraftment i n dogs, and to determine the presence, and percentage, of donor-derive d cells in other canine tissues, a simple and fast method of determini ng cell origin after sex-mismatched bone marrow transplantation was de veloped. Methods. Using universal primers, fragments from genomic DNA corresponding to ZFX and ZFY genes were amplified by polymerase chain reaction. A restriction fragment length polymorphism, combined with de nsitometric analysis, was then used to distinguish and quantitate ZFY and ZFX sequences. Unknown samples were analyzed against standards of known mixtures of male and female DNA. Results. Canine ZFY and ZFX gen es were clearly resolved after amplification, digestion with HaeIII, a nd denaturing polyacrylamide gel electrophoresis. Microchimerism could be detected in male and female dog DNA samples derived from a range o f fresh and frozen tissues including spleen, testicle, and the central nervous system. The levels of chimerism determined using this method were in either agreement with results obtained by karyotyping or more sensitive, with a detection limit of 0.4% compared with 1-2%. Conclusi ons. Polymerase chain reaction/restriction fragment length polymorphis m detection of the ZFY and ZFX genes was found to be simple, accurate, and reliable for assessing engraftment in dogs. When compared with cy togenetic analysis, this method was found to be faster to perform, mor e capable of detecting lower levels of microchimerism, and useful for detecting donor-derived cells in stored specimens and in tissues other than peripheral blood or bone marrow.