Abstract
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. The green florescence proteins (GFP) gene of Aquorea victoria, proved to be a vital marker to identify transformed cells in mixed populations. Use of GFP to observe gene transfer and expression in M. hyorhinis (strain HUB-1) has not been described. We have constructed a pMD18-O/MHRgfp plasmid containing the p97 gene promoter, origin of replication, tetracycline resistance marker and GFP gene controlled by the p97 gene promoter. The plasmid transformed into M. hyorhinis with a frequency of ~4 × 10−3 cfu/µg plasmid DNA and could be detected by PCR amplification of the GFP gene from the total DNA of the transformant mycoplasmas. Analysis of a single clone grown on KM2-Agar containing tetracycline, showed a green fluorescence color. Conclusively, this report suggests the usefulness of GFP to monitor transient gene transfer and expression in M. hyorhinis, eventually minimizing screening procedures for gene transfer and expression.
Highlights
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers
A method based on green florescence proteins (GFP) expressing plasmids to evaluate the transformation and expression of foreign genes in M. hyorhinis has not been described
We previously constructed a plasmid pMD18-TOgfp encoding tetracycline resistance gene controlled by the p97 gene promoter, GFP gene controlled by the p97 gene promoter and oriC of M. hyopneumoniae attenuated strain (168L) (GenBank accession 507382422)
Summary
Mycoplasma hyorhinis (M. hyorhinis) is an opportunistic pathogen of pigs and has been shown to transform cell cultures, which has increased the interest of researchers. A method based on GFP expressing plasmids to evaluate the transformation and expression of foreign genes in M. hyorhinis has not been described. We constructed a plasmid expressing GFP fluorescence and optimized conditions for transformation by electroporation.
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