c-Myc Knockout as a Model for Gene Editing for Training Healthcare Professional Students

Correction of genetic errors, commonly known as gene editing, holds promise to treat diseases with unmet medical needs. However, gene therapy trials do encounter unwanted outcomes, because of an incomplete understanding of the disease states, and gene therapy processes, among others. This situation encourages a concept that healthcare professionals receiving laboratory research training will not only identify inadequacies in basic biomedical knowledge of gene therapies but also provide tangible refinements. To this end, we have undertaken the PharmD student training in gene editing in a basic research laboratory setting. As a model, MYC gene was chosen for knockout using CRISPR-Cas9 method in HT29 and OVCAR8 cells. Students were involved in the design of MYC-specific gRNAs, subcloning into Cas9-carrying plasmid, and selection of knockout clones from the transfected cells. Subsequently, genomic DNA isolation and sequencing, analysis of clonal DNA sequences using online bioinformatics tools, western blotting, cell proliferation and cell division cycle experiments, were performed to characterize the MYC knockout clones. Results presented in this communication suggest that healthcare professionals who received laboratory training gain a better understanding of the disease states and mechanisms, gene therapy protocols, limitations of gene therapies, ability to critically evaluate the literature and confidence in the oversight of gene therapies in the clinic.

CALD1 facilitates epithelial-mesenchymal transition progression in gastric cancer cells by modulating the PI3K-Akt pathway

BACKGROUND CALD1 has been discovered to be abnormally expressed in a variety of malignant tumors,including gastric cancer(GC),and is associated with tumor progression and immune infiltration;however,the roles and mechanisms of CALD1 in epithe-lial-mesenchymal transition(EMT)in GC are unknown.AIM To investigate the role and mechanism of CALD1 in GC progression,invasion,and migration.METHODS In this study,the relationship between CALD1 and GC,as well as the possible network regulatory mechanisms of CALD1,was investigated by bioinformatics and validated by experiments.CALD1-siRNA was synthesized and used to trans-fect GC cells.Cell activity was measured using the CCK-8 method,cell migration and invasive ability were measured using wound healing assay and Transwell assay,and the expression levels of relevant genes and proteins in each group of cells were measured using qRT-PCR and Western blot.A GC cell xenograft model RESULTS Bioinformatics results showed that CALD1 was highly expressed in GC tissues,and CALD1 was significantly higher in EMT-type GC tissues than in tissues of other types of GC.The prognosis of patients with high expression of CALD1 was worse than that of patients with low expression,and a prognostic model was constructed and evaluated.The experimental results were consistent with the results of the bioinformatics analysis.The expression level of CALD1 in GC cell lines was all higher than that in gastric epithelial cell line GES-1,with the strongest expression found in AGS and MKN45 cells.Cell activity was significantly reduced after CALD1-siRNA trans-fection of AGS and MKN45 cells.The ability of AGS and MKN45 cells to migrate and invade was reduced after CALD1-siRNA transfection,and the related mRNA and protein expression was altered.According to bioinfor-matics findings in GC samples,the CALD1 gene was significantly associated with the expression of members of the PI3K-AKT-mTOR signaling pathway as well as the EMT signaling pathway,and was closely related to the PI3K-Akt signaling pathway.Experimental validation revealed that upregulation of CALD1 increased the expression of PI3K,p-AKT,and p-mTOR,members of the PI3K-Akt pathway,while decreasing the expression of PTEN;PI3K-Akt inhibitor treatment decreased the expression of PI3K,p-AKT,and p-mTOR in cells overexpressing CALD1(still higher than that in the normal group),but increased the expression of PTEN(still lower than that in the normal group).CCK-8 results revealed that the effect of CALD1 on tumor cell activity was decreased by the addition of the inhibitor.Scratch and Transwell experiments showed that the effect of CALD1 on tumor cell migration and invasion was weakened by the addition of the PI3K-Akt inhibitor.The mRNA and protein levels of EMT-related genes in AGS and MKN45 cells were greatly altered by the overexpression of CALD1,whereas the effect of overex-pression of CALD1 was significantly weakened by the addition of the PI3K-Akt inhibitor.Animal experiments showed that tumour growth was slow after inhibition of CALD1,and the expression of some PI3K-Akt and EMT pathway proteins was altered.CONCLUSION Increased expression of CALD1 is a key factor in the progression,invasion,and metastasis of GC,which may be associated with regulating the PI3K-Akt pathway to promote EMT.

Establishment of a Genetic Transformation System and Its Application in Thermoanaerobacter tengcongensis

The whole-genome sequence of Thermoanaerobacter tengcongensis, an anaerobic thermophilic bacterium isolated from the Tengchong hot spring in China, was completed in 2002. However, in vivo studies on the genes of this strain have been hindered in the absence of genetic manipulation system. In order to establish such a system, the plasmid pBOL01 containing the replication origin of the T. tengcongensis chromosome and a kanamycin resistance cassette, in which kanamycin resistance gene expression was controlled by the tte1482 promoter from T. tengcongensis, was constructed and introduced into T. tengcongensis via electroporation. Subsequently, the high transformation efficiency occurred when using freshly cultured T. tengcongensis cells without electroporation treatment, suggesting that T. tengcongensis is naturally competent under appropriate growth stage. A genetic transformation system for this strain was then established based on these important components, and this system was proved to be available for studying physiological characters of T. tengcongensis in vivo by means of hisG gene disruption and complementation.

Molecular characterization and m RNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 mRNA expression was found under exposure to thyroid hormone （T4） and estrogen （estradiol）, whereas expression of the corresponding biomarker genes was induced. Our results show that rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.