Reporter mice express green fluorescent protein at initiation of meiosis in spermatocytes

Transgenic mice were generated using a heat shock protein 2 (Hspa2) gene promoter to express green fluorescent protein (GFP) at the beginning of meiotic prophase I in spermatocytes. Expression was confirmed in four lines by in situ fluorescence, immunohistochemistry, western blotting, and PCR assays. The expression and distribution of the GFP and HSPA2 proteins co‐localized in spermatocytes and spermatids in three lines, but GFP expression was variegated in one line (F46), being present in some clones of meiotic and post‐meiotic germ cells and not in others. Fluorescence activated cell sorting (FACS) was used to isolate purified populations of spermatocytes and spermatids. Although bisulfite sequencing revealed differences in the DNA methylation patterns in the promoter regions of the transgene of the variegated expressing GFP line, a uniformly expressing GFP reporter line, and the Hspa2 gene, these differences did not correlate with variegated expression. The Hspa2‐GFP reporter mice provide a novel tool for studies of meiosis by allowing detection of GFP in situ and in isolated spermatogenic cells. They will allow sorting of meiotic and post‐meiotic germ cells for characterization of molecular features and correlation of expression of GFP with stage‐specific spermatogenic cell proteins and developmental events. genesis 52:976–984, 2014. © 2014 Wiley Periodicals, Inc.

[1]  Sarah C R Elgin,et al.  Position-effect variegation, heterochromatin formation, and gene silencing in Drosophila. , 2013, Cold Spring Harbor perspectives in biology.

[2]  M. Griswold,et al.  Retinoic acid regulation of male meiosis , 2013, Current opinion in endocrinology, diabetes, and obesity.

[3]  T. Garcia,et al.  Isolation of undifferentiated and early differentiating type A spermatogonia from Pou5f1-GFP reporter mice. , 2012, Methods in molecular biology.

[4]  A. Geisinger,et al.  Flow Cytometry for Gene Expression Studies in Mammalian Spermatogenesis , 2010, Cytogenetic and Genome Research.

[5]  N. Nakamura,et al.  Heat shock protein 2 promoter drives cre expression in spermatocytes of transgenic mice , 2009, Genesis.

[6]  M. Bao-Cutrona,et al.  Unexpected Expression Pattern of Tetracycline-Regulated Transgenes in Mice , 2009, Genetics.

[7]  M. Barchi,et al.  Isolation and analyses of enriched populations of male mouse germ cells by sedimentation velocity: the centrifugal elutriation. , 2009, Methods in molecular biology.

[8]  M. Handel,et al.  Isolation and short-term culture of mouse spermatocytes for analysis of meiosis. , 2009, Methods in molecular biology.

[9]  D. Kioussis,et al.  Position effect variegation and imprinting of transgenes in lymphocytes , 2008, Nucleic acids research.

[10]  F. DeMayo,et al.  Peroxisome Proliferator-Activated Receptor γ Is a Target of Progesterone Regulation in the Preovulatory Follicles and Controls Ovulation in Mice , 2008, Molecular and Cellular Biology.

[11]  P. Reddi,et al.  A Novel CpG-free Vertebrate Insulator Silences the Testis-specific SP-10 Gene in Somatic Tissues , 2007, Journal of Biological Chemistry.

[12]  R. Brinster,et al.  Culture Conditions and Single Growth Factors Affect Fate Determination of Mouse Spermatogonial Stem Cells1 , 2004, Biology of reproduction.

[13]  M. Griswold,et al.  The Murine Testicular Transcriptome: Characterizing Gene Expression in the Testis During the Progression of Spermatogenesis1 , 2004, Biology of reproduction.

[14]  M. Stoler,et al.  Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells. , 2003, Developmental biology.

[15]  R. Brinster,et al.  Spermatogonial stem cells share some, but not all, phenotypic and functional characteristics with other stem cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. McDonald,et al.  Complex patterns of inheritance of an imprinted murine transgene suggest incomplete germline erasure. , 2000, Nucleic acids research.

[17]  J. Herr,et al.  Round spermatid-specific transcription of the mouse SP-10 gene is mediated by a 294-base pair proximal promoter. , 1999, Biology of reproduction.

[18]  P. Reddi,et al.  Green fluorescent protein as a reporter for promoter analysis of testis-specific genes in transgenic mice. , 1999, Methods in enzymology.

[19]  David Garrick,et al.  Repeat-induced gene silencing in mammals , 1998, Nature Genetics.

[20]  D. Dix,et al.  Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[21]  D. A. O’Brien.  15 – Isolation, Separation, and Short-Term Culture of Spermatogenic Cells , 1993 .

[22]  D. A. O’Brien,et al.  Identification of the gene for the developmentally expressed 70 kDa heat-shock protein (P70) of mouse spermatogenic cells. , 1992, Developmental biology.

[23]  P. Leder,et al.  Parental-specific methylation of an imprinted transgene is established during gametogenesis and progressively changes during embryogenesis , 1991, Cell.

[24]  D. A. O’Brien,et al.  Expression of heat shock proteins by isolated mouse spermatogenic cells , 1988, Molecular and cellular biology.

[25]  D. A. O’Brien,et al.  A novel hsp70-like protein (P70) is present in mouse spermatogenic cells , 1988, Molecular and cellular biology.

[26]  R. Palmiter,et al.  Tissue‐specific, inducible and functional expression of the E alpha d MHC class II gene in transgenic mice. , 1985, The EMBO journal.

[27]  D. A. O’Brien,et al.  Dissociation of the mouse testis and characterization of isolated spermatogenic cells. , 1977, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[28]  M. Meistrich,et al.  Centrifugal elutriation: Separation of spermatogenic cells on the basis of sedimentation velocity , 1975, Journal of cellular physiology.