ABSTRACT
The CRISPR/Cas system is currently widely used for genome editing. The procedure of genome editing includes two necessary steps: (i) searching for the most effective guide RNA, and (ii) analyzing clones for presence of the desired mutation. This review presents the methods used to assess the efficiency of the CRISPR/Cas system and to confirm mutation in the target locus and discusses their advantages and disadvantages. It aims to provide information that could help researchers to choose a technique most appropriate for their specific tasks and available resources.
Subject(s)
CRISPR-Cas Systems/genetics , Gene Editing/methods , Gene Editing/standardsABSTRACT
Understanding of the functioning of MUC1 (human mucin) has advanced significantly over 40 years of its investigation. The anti-adhesive properties of the extracellular domain, which were the main focus of early studies initially explaining overexpression of MUC1 in progressing oncological diseases, were gradually put on the back burner. Researchers became more interested in its regulatory and signaling functions in cells rather in its anti-adhesive properties. The found the ability of MUC1 for signal transduction, and its ability to participate in cell metabolism opened new possibilities for improved control over cancer cells in addition to just attracting antigens of the immune system to a target. Nevertheless, there are issues in the functioning of MUC1 that raise doubts about its effectiveness in cancer immunotherapy.
Subject(s)
Immunosuppressive Agents/metabolism , Immunotherapy , Molecular Targeted Therapy , Mucin-1/chemistry , Mucin-1/metabolism , Neoplasms/therapy , Biomarkers, Tumor/chemistry , Biomarkers, Tumor/metabolism , CD4-Positive T-Lymphocytes/metabolism , Glycosylation , Humans , Immunosuppressive Agents/chemistry , Lymphocyte Activation , Protein Isoforms , Signal Transduction , Tandem Repeat SequencesABSTRACT
Human mucin MUC1 plays an important role in cancer development. The increased level of this molecule expression during cancer cell progression induces metastasis and is associated with poor prognosis for patients. There is a large body of experimental data on the role of various functional domains of human mucin MUC1 in metastasis. While, the cytoplasmic domain determined to play a definitive role, the influence of extracellular domain on cancer cell invasiveness still remains unclear. The present paper reveals that the extracellular domain of MUC1 molecule consists of two functional subdomains-the region of tandem repeats (TR) and the region of irregular repeats (IR). We demonstrate the ability of each of these subdomains to alter the invasiveness of cancer cells. The presence of the MUC1 molecules containing TR subdomain (MUC1-TR) on the surface of low-invasive cancer cells leads to the increase in their transendothelial migration potency, while the addition of the IR subdomain to the MUC1-TR molecule (MUC1-IR-TR) restores their natural low invasiveness. J. Cell. Biochem. 118: 4002-4011, 2017. © 2017 Wiley Periodicals, Inc.
Subject(s)
Cell Movement , Mucin-1/metabolism , Neoplasm Proteins/metabolism , Neoplasms/metabolism , Cell Line, Tumor , Humans , Mucin-1/chemistry , Mucin-1/genetics , Neoplasm Invasiveness , Neoplasm Metastasis , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Neoplasms/chemistry , Neoplasms/genetics , Protein DomainsABSTRACT
The speculations on the role of MUC1, a substance which is overexpressed in glandular cancer cells, on the metastatic potential of such cells are rooted in data that seem to indicate that cell malignization correlates with a change from the apical localization of mucin MUC1 to a peripheral one. Nonetheless, the role of MUC1 in cancer metastasizing remains far from clear. The major hurdle remains the absence of adequate cell models. The aim of the present study was to create cell models that present different fragments of the human mucin MUC1 extracellular domain on their surface. Genetic constructions were generated on the basis of the plasmid vector pEGFP-N3. These constructions contain fusion genes coding for chimeric proteins composed of different combinations of mucin MUC1 functional domains and identification markers (FLAG-epitope, located at the N-terminus, and EGFP, located at the C-terminus of the chimeric proteins). These constructions were used for a stable transformation of HT-29 human cancer cells. The transformants obtained were characterized by flow cytometry. The low expression level of endogenous mucin MUC1 and the high expression level of recombinant proteins were confirmed by real-time PCR. The microscopic examination of the transformed cells confirmed the membrane localization of the fusion proteins. The resulting cell models could be used to investigate the role of the mucin MUC1 domains in cancer cell metastasizing. The obtained cells are used as an applicable model of MUC1-expressing cancers and might be used to study the role of different functional fragments of mucin MUC1 in metastasizing.
