A new method is described for the purification of spermatogenic cell populations from mouse testis. Through use of this method, it is possible to purify leptotene, zygotene, and pachytene primary spermatocytes as well as round spermatids from adult mouse testis. In addition, spermatogonial populations can be purified from mice at 9 days postpartum. The leptotene and zygotene primary spermatocytes that can be prepared by this method are impossible to separate successfully by the unit gravity method. The cells were used to prepare RNA for reverse transcriptase-polymerase chain reactions.
Sequences of DNA that hybridize on Southern blots with cloned EcoR1 1.3 kb (ER1) of long interspersed repeated sequence (L1Md) of mouse have been examined in genomic DNA of neonatal mice, livers and brains of adult mice (3, 10, 27, and 30 mo old), and the solid myeloma tumor MOPC-315. The isoschizomers Hpa II (CCGG or mCCGG) and Msp I (CCGG or CmCGG) were used to assess methylation. We found that the L1Md sequence is fully methylated in young animals but demethylated in myeloma. Demethylation of L1Md sequence also occurred in aged animals. By scanning the autoradiogram, we found that approximately 8% of the 10(4)-10(5) copies have been demethylated in 27-mo-old liver.
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