CRISPR-Cas9 mediated capsule gene silencing in Escherichia coli

Background: Genomic engineering of Escherichia coli is applied to design and produce recombinant proteins as the new drugs. The aim of this study was to CRISPR-Cas9 mediated capsule gene silencing in E. coli

Materials and Methods: We suppressed genes involved in capsule expression of E.coli by CRISPR cas9 process. The constructed E.coli was confirmed by microscopic smear, transmission electron microscopy and T7 phage influence assay

Results: The results were shown that the inhibition of capsule production was carried out successfully and there was not any capsule layer around the bacteria

Conclusion: E. coli without any capsule around may proper for replacement of it with other molecules in future

Development of flow cytometry-fluorescent in situ hybridization [Flow-FISH] method for detection of PML/RARa chromosomal translocation in acute promyelocytic leukemia cell line

Background: acute Promyelocytic Leukemia [APL] is a subclass of acute myeloid leukemia. The chromosomal aberration in 95% of APL cases is t[15; 17] [q22; q21], which prevents cell differentiation. Characterization of the underlying molecular lesion is valuable in determining optimal treatment strategy. The goal of this study was to develop a new and powerful Flow- FISH technique to detect the long isoform [L] of PML-RARa fusion transcript in NB4 cell line

Methods: to achieve the best condition for fixation, two different fixatives including 2% paraformaldehyde and 75% ethanol were used. 0.2% Triton X-100 and 0.2% saponin were used for the permeabilization step .In hybridization, a wide range of times and temperatures were used and probe was designed in FRET system. Results were confirmed by fluorescent microscope assay and reverse transcription PCR

Results: in the present study, a novel technique was successfully optimized that combines in situ hybridization with flow cytometry to detect the presence of PML-RARa transcript. Using standard fixation and permeabilization protocol of 2% PFA and 0.2% saponin gave the best fluorescent results in flow cytometry. Also, results indicated that the optimum time and temperature for hybridization was 2 hr at 42degreeC. The results of reverse transcription PCR and fluorescent microscopy confirmed the presence of PML-RARa transcript

Conclusion: the concordance between the results of Flow-FISH and those of two other techniques including reverse transcription PCR and FISH indicated that this method would be applicable as a diagnostic test for APL in clinical samples and MRD monitoring