engleski

Transgenic Stra8 promoter activity in mice

Our approach was to identify a transcriptional promoter which could address gene expression to the early stage of germ cell development. No promoter so far has been described with that unique specificity. The Stra8 gene was initially identify as a retinoid regulated gene expressed in P19 cells. In vivo it was found to be expressed in mice spermatogonia so 400 pb of the Stra8 promoter was used in the construct in order to isolate pure spermatogonia. First construct was made with the luciferase as a reporter gene and second construct with the neutral surface marker. The original human CD4 molecule was modified in the transmembrane and intracytoplasmic regions and was fused with two extracellular domains. This modified marker allowed the recognition of the cells and isolation with the CD4 antibody coupled with magnetic beads. In the extracts of all tissues of the trangenic male and female mice carrying Stra8/luc construct the luciferase activity was measured in luminometer. The highest activity was administred in testis, small activity in the brain while in the other tissues of the male and female mice there were no activity. Immunohistochemistry with the anti-luciferase antibody showed the expression in the cells of the newborn testis. In the adult testis the luciferase expression was shown in the cells situated on the basal membrane. Endogenous Stra8 expression was administred in all cells on the basal membrane that correspond to the spermatogonia and perhaps early spermatocytes. Transgenic expression of the luciferase was administred in just few cells that could correspond to the subpopulation of the spermatogonia. This construct was made to see the distribution of the expresion in the mice and it's specifity. The result showed that the 400 pb of the Stra8 promoter was enought for the espression of the reporter gene and was testis specific and was expressed in the subpopulation of the cells on the basal membrane. In the Stra8/CD4 transgenic mice the modified CD4 molecule was expressed under the Stra8 promoter and the cells were recognised with Dynabeads CD4.

transgenic; spermatogonia; promoter; Stra8

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