Triplet repeats and DNA repair: germ cell and somatic cell instability in transgenic mice.
This chapter describes methods for the isolation of specific cell types that reveal how and where expansion can occur. For the hereditary component of expansion, the male germ cell has proved useful in distinguishing processes that can contribute to expansion, as described in our article (Nature Genetics 27, 407, 2001). Mature spermatazoa (SZs) can be isolated directly from the epididymis. Haploid spermatids (STs), diploid spermatagonia (SGs), and tetraploid spermatocytes (SCs) can be removed from the testis and sorted by fluorescence-activity cell sorting (FACS); differences in DNA content and morphology allow resolution by fluorescence and light scattering. Repeat-length measurement can pinpoint the stage at which expansion occurs. Because the timing of meiosis and mitosis with respect to sperm development is known, the analysis can distinguish repair and replication processes. Furthermore, the possible contribution of Y- or X-specific factors can be evaluated by sorting X- and Y-bearing germ cells. To enable analysis of female germ cells, we describe methods for oocyte preparations and a method for the isolation of the eight-cell-stage embryo. Therefore, the methods described here can help to answer such questions as the timing during development of expansion, whether expansion is limited to a single period, whether both replication and repair contribute to instability, and the role of somatic instability in disease. If further expansion of the inherited allele contributes to the phenotype, then intervention in somatic tissue might be therapeutic.
Kovtun
Irina V
IV
Department of Pharmacology, Mayo Clinic, Rochester, MN, USA.
Spiro
Craig
C
McMurray
Cynthia T
CT
D000818:Animals; D004260:DNA Repair / Q000235:genetics*; D005260:Female; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D013094:Spermatozoa / Q000166:cytology; D018911:Trinucleotide Repeats