| Popis: |
In order to follow the course of bone marrow engraftment in dogs, and to determine the presence, and percentage, of donor-derived cells in other canine tissues, a simple and fast method of determining cell origin after sex-mismatched bone marrow transplantation was developed.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 densitometric 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.Canine ZFY and ZFX genes were clearly resolved after amplification, digestion with HaeIII, and denaturing polyacrylamide gel electrophoresis. Microchimerism could be detected in male and female dog DNA samples derived from a range of 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%.Polymerase chain reaction/restriction fragment length polymorphism detection of the ZFY and ZFX genes was found to be simple, accurate, and reliable for assessing engraftment in dogs. When compared with cytogenetic analysis, this method was found to be faster to perform, more 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. |
