Identification of germ-line chimaeras by polymerase chain reaction and isoenzyme analysis of mouse spermatozoa.
In this study a rapid, simple and inexpensive procedure is described which allows potential germ-line male mice to be identified with confidence. Spermatozoa recovered by uterine washing following mating with normal female mice was analysed in two ways. First, the patterns of expression of the different isoforms of glucose phosphate isomerase were determined. Since the glucose phosphate isomerase isoforms expressed in embryo stem (ES) cell lines are frequently different from those associated with the host blastocyst, it is possible to determine the proportion of spermatozoa produced by an individual animal that was of ES cell or host-blastocyst origin. Second, DNA of spermatozoa was subjected to polymerase chain reaction (PCR) analysis using primers with specificity for the targeted mutation in the ES cells. The PCR analysis was particularly valuable in identifying germ cell chimaeras in which the contribution of ES-derived spermatozoa was significantly less than that specified by the host blastocyst.
Mann
G B
GB
Melbourne Tumour Biology Branch, Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Victoria, Australia.
Fowler
K J
KJ
Grail
D
D
Dunn
A R
AR
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002678:Chimera; D004247:DNA / Q000235:genetics; D005956:Glucose-6-Phosphate Isomerase / Q000032:analysis*; D007527:Isoenzymes; D008297:Male; D051379:Mice; D008815:Mice, Inbred Strains; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010641:Phenotype; D016133:Polymerase Chain Reaction; D013094:Spermatozoa / Q000201:enzymology* / Q000502:physiology; D013234:Stem Cells / Q000201:enzymology*
D007527:Isoenzymes; D004247:DNA; D005956:Glucose-6-Phosphate Isomerase