Preparation and Use of shRNA for Knocking Down Specific Genes.

Short hairpin RNA (shRNA) is a technique used to silence gene expression stably in various cells. There are however several reported problems. First, the cloning of oligos can lead to ligation of multiple copies; second, premature termination of sequencing reaction during confirmation of hairpin template; third, microdeletions/substitutions in hairpin during cloning; and fourth, off target effects.

Site-Directed Mutagenesis to Mutate Multiple Residues in a Single Reaction.

Site-directed mutagenesis (SDM) is a technique that allows mutation of specific nucleotide(s) in a codon to study its functional implications in a protein. Commercial kits are available, which require high-performance liquid chromatography purified oligos for this purpose. These kits are expensive, and they are not very efficient, so one has to sequence several clones to get a desired one.

Transcriptome Analysis Reveals Vimentin-Induced Disruption of Cell-Cell Associations Augments Breast Cancer Cell Migration.

In advanced metastatic cancers with reduced patient survival and poor prognosis, expression of vimentin, a type III intermediate filament protein is frequently observed. Vimentin appears to suppress epithelial characteristics and augments cell migration but the molecular basis for these changes is not well understood. Here, we have ectopically expressed vimentin in MCF-7 and investigated its genomic and functional implications.

Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly.

Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging.

Major Molecular Signaling Pathways in Oral Cancer Associated With Therapeutic Resistance.

Oral cancer is a sub-category of head and neck cancers that primarily initiates in the oral cavity. The primary treatment option for oral cancer remains surgery but it is associated with massive disfigurement, inability to carry out normal oral functions, psycho-social stress and exhaustive rehabilitation. Other treatment options such as chemotherapy and radiotherapy have their own limitations in terms of toxicity, intolerance and therapeutic resistance.

Vimentin Is at the Heart of Epithelial Mesenchymal Transition (EMT) Mediated Metastasis.

Epithelial-mesenchymal transition (EMT) is a reversible plethora of molecular events where epithelial cells gain the phenotype of mesenchymal cells to invade the surrounding tissues. EMT is a physiological event during embryogenesis (type I) but also happens during fibrosis (type II) and cancer metastasis (type III). It is a multifaceted phenomenon governed by the activation of genes associated with cell migration, extracellular matrix degradation, DNA repair, and angiogenesis.

Mutations in SPATA13/ASEF2 cause primary angle closure glaucoma.

Current estimates suggest 50% of glaucoma blindness worldwide is caused by primary angle-closure glaucoma (PACG) but the causative gene is not known. We used genetic linkage and whole genome sequencing to identify Spermatogenesis Associated Protein 13, SPATA13 (NM_001166271; NP_001159743, SPATA13 isoform I), also known as ASEF2 (Adenomatous polyposis coli-stimulated guanine nucleotide exchange factor 2), as the causal gene for PACG in a large seven-generation white British family showing variable expression and incomplete penetrance. The 9 bp deletion, c.

Serum lipids, retinoic acid and phenol red differentially regulate expression of keratins K1, K10 and K2 in cultured keratinocytes.

Abnormal keratinocyte differentiation is fundamental to pathologies such as skin cancer and mucosal inflammatory diseases. The ability to grow keratinocytes in vitro allows the study of differentiation however any translational value is limited if keratinocytes get altered by the culture method. Although serum lipids (SLPs) and phenol red (PR) are ubiquitous components of culture media their effect on differentiation is largely unknown.

Hepatitis C in Pakistan: a review of available data.

Hepatitis C virus (HCV) infection is increasingly recognized as a major health care problem, and is found frequently in Pakistani settings. In this article we reviewed published and unpublished data related to the seroepidemiology of HCV infection in Pakistan. For this article, data from 132 published studies and three unpublished data sets published/ presented between the period 1992-2008 were utilized.