Detection of meiotic DNA breaks in mouse testicular germ cells.
The study of location and intensity of double-strand breaks (DSBs) in mammalian systems is more challenging than in yeast because, unlike yeast, the progression through meiosis is not synchronous and only a small fraction of all testis cells are actually at the stage where DSB formation is initiated. We devised a quantitative approach that is sensitive enough to detect the position of rare DNA strand breaks in mouse germ cell-enriched testicular cell populations. The method can detect DNA breaks at any desired location in the genome but is not specific for DSBs-overhangs, nicks, or gaps with a free 3' OH group are also detected. The method was successfully used to compare testicular cells from mouse strains that possess or lack an active recombination hot spot at the H2-Ea gene. Breaks that were due to meiotic hot spot activity could be distinguished from the background of DNA breaks. This highly sensitive approach could be used to study other biological processes where rare DNA breaks are generated.
Fluidigm Corporation, South San Francisco, CA, USA.
Jian
J
Qin
Jaichandar
J
Subramanian
Norman
N
Arnheim
D000818:Animals; D001483:Base Sequence; D053903:DNA Breaks, Double-Stranded; D004252:DNA Mutational Analysis / Q000379:methods*; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism*; D008297:Male; D051379:Mice; D008954:Models, Biological; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping / Q000379:methods; D013094:Spermatozoa / Q000166:cytology / Q000378:metabolism; D013737:Testis / Q000166:cytology / Q000378:metabolism*