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First published online 17 August 2005
doi: 10.1242/dev.02004

Development 132, 4155-4163 (2005)
Published by The Company of Biologists 2005
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Genetic and epigenetic properties of mouse male germline stem cells during long-term culture

Mito Kanatsu-Shinohara1,2, Narumi Ogonuki3, Tomohiko Iwano4, Jiyoung Lee2,5, Yasuhiro Kazuki6, Kimiko Inoue3, Hiromi Miki3, Masanori Takehashi2, Shinya Toyokuni7, Yoichi Shinkai4, Mitsuo Oshimura6, Fumitoshi Ishino5, Atsuo Ogura3 and Takashi Shinohara2,*

1 Horizontal Medical Research Organization, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
2 Department of Molecular Genetics Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan
3 RIKEN, Bioresource Center, Ibaraki 305-0074, Japan
4 Experimental Research Center for Infectious Diseases, Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
5 Medical Research Institute, Tokyo Medical and Dental University, Tokyo 101-0062, Japan
6 Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan
7 Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan



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  		Fig. 1. Phenotypic analysis of GS cells. (A) Left, appearance of GS cells, 24 months after initiation of the cultures; middle, alkaline phosphatase activity in the GS cells; right, appearance of mGS cells that closely resemble ES cells. (B) Flow cytometric characterization of GS cells after 24 months in culture. Black line, control immunoglobulin; red line, specific antibody. (C) RT-PCR analysis. Specific primers were used to amplify cDNA from GS and mGS cells. (D) Karyotype analysis of two separate cultures of GS cells. At least 50 cells were counted. (E) Cumulative growth curves for two separate cultures of GS cells. Cells were maintained for 2 years in which the total number of cells at each passage has been calculated. There is steady and consistent exponential increase in total cell number over time. Scale bar: 100 µm in A.

 


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  		Fig. 2. Analysis of telomere dynamics and telomerase activity. (A) Southern blot analysis of TRFs containing telomeric DNA sequences in five different ES (E14) cells and GS cells. Genomic DNAs from ES or GS cells were digested with HinfI, separated on a pulse-field gel and probed with the 5'-[32P](T2AG3)3 telomeric DNA oligonucleotides. (B) Loss of telomeres in GS cells. Based on the median values of the TRF analysis shown in (A), shortening rates of the telomeric DNA sequences in GS cells were calculated for two separate cultures. (C) Telomerase activity in ES and GS cells. ES and GS cells were subjected to a standard telomeric repeat amplification assay. Extracts were pretreated with (H) or without heat inactivation (80°C, 10 minutes) prior to the telomerase assay. The total amount of cell lysates per assay is indicated.

 


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  		Fig. 3. COBRA of imprinted genes. PCR products of each DMR region were digested with restriction enzymes with a recognition sequence containing CpG in the original unconverted DNA. Where the enzyme sites are methylated, PCR products are fragmented; where PCR products are not fragmented, the enzyme sites are unmethylated. White arrowheads indicate the sizes of the unmethylated DNA fragments. Black arrowheads indicate the sizes of the methylated DNA fragments. The enzymes used to cleave each locus are indicated in parenthesis. BsiEI was used to analyze the Rasgrf1 DMRs in the DNA from mouse pups and from mGS cells. Levels of percentage methylation, estimated by the intensity of each band, are indicated below the gels. U, uncleaved; C, cleaved.

 


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  		Fig. 4. Spermatogenesis and the generation of offspring from GS cells. EGFP-expressing GS cells from 24-month-old cultures were transplanted into the seminiferous tubules of infertile W recipient mice. (A) A recipient testis showing the EGFP fluorescence in GS cell-derived colonies. Green tubules represent donor GS cell colonization. (B) Histological analysis (Hematoxylin and Eosin staining) of the recipient testis showing normal-appearing spermatogenesis. (C) Round spermatid released from a recipient testis, exhibiting EGFP fluorescence (arrow). (D) Offspring resulting from the microinjection of oocytes with round spermatids, exhibiting fluorescence. Scale bar: 1 mm.

 

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© The Company of Biologists Ltd 2005