Short-homology-mediated PCR-based method for gene introduction in the fission yeast Schizosaccharomyces pombe.

Schizosaccharomyces pombe is a commonly utilized model organism for studying various aspects of eukaryotic cell physiology. One reason for its widespread use as an experimental system is the ease of genetic manipulations, leveraging the natural homology-targeted repair mechanism to accurately modify the genome. We conducted a study to assess the feasibility and efficiency of directly introducing exogenous genes into the fission yeast S. pombe using Polymerase Chain Reaction (PCR) with short-homology flanking sequences. Specifically, we amplified the NatMX6 gene (which provides resistance to nourseothricin) using PCR with oligonucleotides that had short flanking regions of 20 bp, 40 bp, 60 bp and 80 bp to the target gene. By using this purified PCR product, we successfully introduced the NatMX6 gene at position 171 385 on chromosome III in S. pombe. We have made a simple modification to the transformation procedure, resulting in a significant increase in transformation efficiency by at least 5-fold. The success rate of gene integration at the target position varied between 20% and 50% depending on the length of the flanking regions. Additionally, we discovered that the addition of dimethyl sulfoxide and boiled carrier DNA increased the number of transformants by ~60- and 3-fold, respectively. Furthermore, we found that the removal of the pku70+ gene improved the transformation efficiency to ~5% and reduced the formation of small background colonies. Overall, our results demonstrate that with this modified method, even very short stretches of homologous regions (as short as 20 bp) can be used to effectively target genes at a high frequency in S. pombe. This finding greatly facilitates the introduction of exogenous genes in this organism.

A CD44 specific peptide developed by phage display for targeting gastric cancer.

OBJECTIVE: To develop a peptide probe that could be used for gastric cancer detection via binding to CD44 protein with specificity and affinity. RESULTS: A 12-mer phage peptide library was screened against immobilized CD44 protein. Bound phage counts using ELISA were performed to identify phage clones carrying the most highly selective peptide, which termed RP-1. Immunofluorescence and flow cytometry analysis indicated that the consensus peptide RP-1 could bind to CD44-positive gastric cancer cells with mean fluorescence intensities significantly higher than that of CD44-negative cells. CD44 knockdown led to decreased binding activity of RP-1 to the same cell line. Tissue array technique was used to identify the relationship (r = 0.556) between peptide binding and CD44 detection on gastric cancer tissues. Further, the hyaluronan-binding domain of CD44 was docked with RP-1 using computer modeling/docking approaches, revealing a RP-1/CD44 interaction with geometrical and energy match (-8.6 kcal/mol). CONCLUSIONS: The RP-1 peptide we screened exhibits affinity and specificity to CD44 on cells and has the potential to be used as a candidate probe for gastric cancer cell targeting.

ANXA2 regulates the behavior of SGC-7901 cells.

ANXA2, a member of the annexin family, is overexpressed and plays important roles in tumor development. However, the significance of ANXA2 expression in gastric carcinoma has not been clarified.To elucidate its roles in growth of gastric cancer, ANXA2 expression in SGC-7901 cells was inhibited with a designated siRNA, then cell proliferation, cell cycling, apoptosis and motility were determined by MTT assay, flow cytometry, Hoechst 33342 staining and wound healing assay, respectively. To further assess the behavior of ANXA2 deleted SGC- 7901 cells, changes of microstructures were observed under fluorescence microscopy, laser scanning confocal microscopy and electron microscopy. We found that inhibition of ANXA2 expression caused cell proliferation to decrease significantly with G1 arrest, motility to be reduced with changes in pseudopodia/filopodia structure and F-actin and ß-tubulin expression, and apoptosis to be enhanced albeit without significance. At the same time, ANXA2 deletion resulted in fewer pseudopodia/filopodia, non-stained areas were increased, contact inhibition among cells reappeared, and expression of F-actin and ß-tubulin was decreased, with induction of polymerized disassembled forms. Taken together, these data suggest that ANXA2 overexpression is important to maintain the malignancy of cancer cells, and this member of the annexin family has potential to be considered as a target for the gene therapy of gastric carcinoma.

Affinity peptide developed by phage display selection for targeting gastric cancer.

AIM: To develop an affinity peptide that binds to gastric cancer used for the detection of early gastric cancer. METHODS: A peptide screen was performed by biopanning the PhD-12 phage display library, clearing non-specific binders against tumor-adjacent normal appearing gastric mucosa and obtaining selective binding against freshly harvested gastric cancer tissues. Tumor-targeted binding of selected peptides was confirmed by bound phage counts, enzyme-linked immunosorbent assay, competitive inhibition, fluorescence microscopy and semi-quantitative analysis on immunohistochemistry using different types of cancer tissues. RESULTS: Approximately 92.8% of the non-specific phage clones were subtracted from the original phage library after two rounds of biopanning against normal- appearing gastric mucosa. After the third round of positive screening, the peptide sequence AADNAKTKSFPV (AAD) appeared in 25% (12/48) of the analyzed phages. For the control peptide, these values were 6.8 ± 2.3, 5.1 ± 1.7, 3.5 ± 2.1, 4.6 ± 1.9 and 1.1 ± 0.5, respectively. The values for AAD peptide were statistically significant (P < 0.01) for gastric cancer as compared with other histological classifications and control peptide. CONCLUSION: A novel peptide is discovered to have a specific binding activity to gastric cancer, and can be used to distinguish neoplastic from normal gastric mucosa, demonstrating the potential for early cancer detection on endoscopy.

[Effects of FAK expression level on proliferation and motility of colorectal carcinoma cells].

AIM: To investigate the effect of siRNA targeting FAK gene on proliferation and motility of colorectal cancer. METHODS: Recombinant plasmids that produced siRNAs targeting FAK were designed and cloned, then transfected into Caco-2 cells. The changes of FAK expression levels were examined by RT-PCR and immunocytochemistry. The effects of FAK gene knockdown on apoptotic morphological changes, proliferation, and motility were investigated. RESULTS: Recombinant plasmids targeting FAK were successfully constructed, FAK mRNA and protein level was silenced in Caco-2 cells significantly. The inhibition of mRNA level was achieved maximal at 48 h post transfection with time dependent. The ability of proliferation and motility of Caco-2 cells were significantly decreased. CONCLUSION: Plasmid-mediated FAK siRNA could inhibit FAK gene expression. The proliferation, motility and apoptosis were inhibited effectively, which suggested that FAK expression is closely associated with the proliferation, motility and apoptosis, etc. The results may be used as the reference for gene therapy of colorectal cancer.