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Sox4, a member of the SoxC subfamily which of the Sox family, plays important roles in the development of the vertebrate gonad and nervous system. We have cloned a Sox4 homologue from the brain of Misgurnus anguillicaudatus using homologous cloning and rapid amplification of cDNA ends. We named the cloned gene as MaSox4. The full-length cDNA was 2122 bp, containing a 718 bp 5′-untranslated region and a 267 bp 3′-untranslated region. The open-reading frame of the cloned gene encoded 378 amino acids and contained a characteristic HMG-box DNA-binding domain with the specific motif (RPMNAFMVW). Phylogenetic analysis indicated that MaSox4 is highly homologous to Sox4 in different species. Protein sequence analysis showed that MaSox4 is a nonsecretory hydrophilic protein. Quantitative real-time reverse transcription polymerase chain reaction and in situ hybridization assay revealed that MaSox4 was ubiquitously expressed during embryogenesis and is present in various adult tissues, especially in the central nervous system. Our study suggests that MaSox4 is highly conserved among vertebrates’ evolution and might be involved in developmental processes such as embryogenesis, neurogenesis and gonad development. [ABSTRACT FROM AUTHOR]

Sox7, Sox17 and Sox18 are the members of the Sry-related high-mobility group box family (SoxF) of transcription factors. SoxF factors regulate endothelial cell fate as well as development and differentiation of blood cells and lymphatic vessels. There is very less information about the functions of these genes in fish. We obtained the full-length cDNA sequence of SoxF genes including Sox7, Sox17 and Sox18 in Cyprinus carpio, where Sox7 and Sox18 had two copies. The construction of a phylogenetic tree showed that these genes were homologous to the genes in other species. Chromosome synteny analysis indicated that the gene order of Sox7 and Sox18 was highly conserved in fish. However, immense change in genomic sequences around Sox17 had taken place. Numerous putative transcription factor binding sites were identified in the 5′ flanking regions of SoxF genes which may be involved in the regulation of the nervous system, vascular epidermal differentiation and embryonic development. The expression levels of SoxF genes were highest in gastrula, and was abundantly expressed in the adult brain. We investigated the expression levels of SoxF genes in five specific parts of the brain. The expression levels of Sox7 and Sox18 were highest in the mesencephalon, while the expression level of Sox17 was highest in the epencephalon. In carp, the expression patterns of SoxF genes indicated a potential function of these genes in neurogenesis and in vascular development. These results provide new information for further studies on the potential functions of SoxF genes in carp. [ABSTRACT FROM AUTHOR]

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a well-known method to quantify gene expression by comparing with the reference genes. Generally, housekeeping genes were set as references, as for their stable expression in varying conditions. Here, we try to evaluate few of such genes to identify suitable housekeeping genes as references for qRT-PCR analysis of gene expression in Misgurnus anguillicaudatus. This study evaluated the expression of four commonly used housekeeping genes, i.e. b-actin (ACTB), elongation factor 1 alpha (EF-1a), glyceraldehyde-3-phosphate (GAPDH) and 18S ribosomal RNA (18S rRNA), in gender difference, effects of tissue type, different developmental stages, chemical treatment of embryos/larvae with commonly used vehicles for administration and agents that represent known environmental toxicant. Rank ordering of expression stability was done using geNorm, NormFinder and BestKeeper algorithms. Results suggested that in the qRTPCR test, in all the experimental conditions, EF-1a could be selected as reference gene when analysing a target gene. For the study of different development stages, ACTB could be a candidate as reference gene. For the studies associated with different gender and tissue types, EF-1a would be better targeted as reference gene. Meanwhile, in toxicant treatment, expression of EF-1a seems to be more stable than others and could be considered as reference gene. This study could provide useful guidelines that can be expected to aid M. anguillicaudatus researchers in their initial choice of housekeeping genes for future studies and enable more accurate normalization of gene expression data.