engleski

Purification and characterization of spermatogonial cells expressing neutral surface marker directed by Stra8 promotor gene

Interest in spermatogonia has grown in recent years as a result of exciting developments in stem cell research in general and the development of new research tools. The availability of isolation and culture technique of pure spermatogonia will pave the way for innovative research into stem cell biology, leading to further breakthroughs in understanding of spermatogonial physiology and the development of powerful tools. Our approach was to identify a promoter which in transgenic mice would address gene expression to the most early stages of germ line development. A method was devised for the purification of the cells in which the 400 pb Stra8 promoter was active. It was based on the expression of a protein on the cell surface which does not interfere with cellular function and was recognised by specific antibodies. Stra8/luc and Stra8/HagloCD4 mice transgenic families were established. Suspension of the cells isolated from Stra8/Haglo transgenic mice were prepared and incubated with magnetizable polystyrene beads coated with a primary monoclonal antibody specific for the CD4 antigen. Positive cells after magnetic sorting were recovered from the beads and provided a morphological homogenous fraction. Stem cell characteristics were evaluated by immunocitochemistry with anti-integrin, anti-RBM and anti-Ep-CAM antibodies. Many studies of spermatogonia have been performed with morphological methods but the technique of spermatogonial transplantation developed in 1994 made it possible to study functional aspect of spermatogonial stem cells. In stem cell functional assay based on the ability to repopulate a germ cell depleted testis, CD4 positive cells efficiently generated colonies of differentiating spermatogenic cells.

spermatogonia; promoter; Stra8; isolation

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