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BackgroundThe commonly used laboratory rat, Rattus norvegicus, is unique in having multiple Sry gene copies found on the Y chromosome, with different copies encoding amino acid variations that influence the resulting protein function. It is not clear which Sry genes are expressed at the onset of testis differentiation or how their expression correlates with that of other genes in testis-determination pathways.MethodsHere, two independent E11-E14 developmental RNAseq datasets show that multiple Sry genes are expressed at E12-E13.ResultsThe identified copies expressed during testis initiation include Sry4A, Sry1, and Sry3C, which are conserved in every strain of Rattus norvegicus with genomes sequenced to date.ConclusionsThis work represents a first step in defining the complex environment of rat testis differentiation that can open the door for generating sex reversal model systems using embryo manipulation techniques that have been available in the mouse but not the rat.

2:45 In mammals, testis determination is initiated by expression of the Sty gene. Sry, which is located on the non-recombining region of the Y chromosome, usually exists as a single [...]

Sry is a gene on the Y chromosome that is responsible for testes formation during development. The Sry protein acts as a transcription factor. Sry is also expressed in adult male tissues, which suggests the possibility of additional functions. There are six unique Sry loci present in Rattus norvegicus, and the functions of these are yet to be identified. The objective of this project was to insert each of the six rat Sry genes into the pIVEX2.4 expression plasmid and collect the recombinant protein for use in future Sry research. An N-terminal histidine tag encoded by the pIVEX2.4 vector allowed for one-step purification of the Sry protein by using Ni sepharose 6 fast flow beads (GE Healthcare). All six rat Sry genes were successfully cloned into pIVEX2.4. All constructs were transformed into Lucigen BL21(DE) E. coli and induced for 2 hours with .75mM IPTG at a cell density of 0.8 at OD600. Collection and subsequent analysis of recombinant protein by SDS-PAGE confirmed expression from Sry2/pIVEX2.4A and Sry3/pIVEX2.4A clones. These two Sry proteins are currently being used in gel shift assays to study its role as a transcription factor. [ABSTRACT FROM AUTHOR]

The Sry gene encodes a transcription factor that is a member of the HMG-box family of DNA binding proteins. It plays a vital role in testis determination but is also expressed in adult male tissues. While most mammalian Y chromosomes have only 1 Sry locus, our laboratory has determined that the rat species, Rattus norvegicus, has 6 unique loci. Our objective was to obtain recombinant Sry protein for studies of Sry protein characterization. To achieve this, we prepared stably transfected lines of CHO (Chinese hamster ovarian) cells containing Sry copies 1, 2 and 3. Each of the three types of Sry-transfected cells was maintained in separate 96 well plates with selective media. After two cell passages, the most robust cells from 24 of the wells were selected from each 96 well plate and these new cells were cultured. After 30 days, all cells were collected for screening by an immunoblot. The cell lysates obtained from CHO lines transfected with Sry 3 and analyzed by a western blot indicated that all 24 colony forming units were expressing Sry 3 protein. From this analysis we can conclude that the stably transfected lines of CHO cells containing Sry 3 are successfully incorporating the recombinant DNA and are expressing the desired protein. These cell lines can continue to be used as a source of Sry protein and they also provide a better model of comparison for in vivo expression of Sry than a transiently transfected cell line. [ABSTRACT FROM AUTHOR]