Genome editing in K562 cells suggests a functional role for the XmnI Gg polymorphism: a widely used genetic marker in β-thalassemia and sickle cell disease patients.

The XmnI Gg -158 C/T polymorphism has been widely associated with fetal hemoglobin (HbF) levels, the severity of disease, and the response to the drug hydroxyurea (HU) in both β-thalassemia (β-thal) and sickle cell disease (SCD) patients. However, the functional significance of this single nucleotide polymorphism (SNP) remains unclear. To gain insight, green fluorescence protein (GFP) cassettes harboring the XmnI C or T alleles in their left homology arms (i.

Detection of CRISPR/Cas9-Mediated Fetal Hemoglobin Reactivation in Erythroblasts Derived from Cord Blood-Hematopoietic Stem Cells.

Reactivation of the fetal hemoglobin (HbF) in adult erythroid cells via genome editing is a strategy for the treatment of β-thalassemia and sickle cell disease. In related reports, the reactivation of HbF is regularly examined in erythroblasts which are generated from the adult CD34 hematopoietic stem and progenitor cells (HSPCs). However, the procurement of adult HSPCs, either from the bone-marrow (BM) or from mobilized peripheral-blood (mPB), is difficult.

Pitfalls of Restriction Enzyme Mapping Following Generation of CRISPR Constructs.

Background: The PX330 and the related PX459 plasmids are widely used for Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9-mediated genome editing. Screening for plasmids containing the correct sgRNA template insertion is one of the most important steps in this system. Different methods for screening the sgRNA inserts have been deployed.

Recent advances in CRISPR/Cas9-mediated knock-ins in mammalian cells.

Since its inception, the CRISPR/Cas9 technology has been widely utilized for the targeted insertion of donor DNAs into mammalian genomes. A shortcoming with the earlier knock-in (KI) approaches, however, has been the low efficiency of targeted integrations-especially in primary cells and mouse embryos. Since, a variety of novel strategies have been developed towards improving the KI efficiencies in select target cells.

Efficient Production of Biallelic RAG1 Knockout Mouse Embryonic Stem Cell Using CRISPR/Cas9.

Background: Recombination Activating Genes (RAG) mutated embryonic stem cells are (ES) cells which are unable to perform V (D) J recombination. These cells can be used for generation of immunodeficient mouse. Creating biallelic mutations by CRISPR/Cas9 genome editing has emerged as a powerful technique to generate site-specific mutations in different sequences.

CRISPR/Cas9 System for Efficient Genome Editing and Targeting in the Mouse NIH/3T3 Cells.

Background: The Clustered, Regularly Interspaced, Short Palindromic Repeats (CRIS-PR) and CRISPR-associated protein (Cas) system has been used as a powerful tool for genome engineering. In this study, the application of this system is reported for targeting genes to produce mutant mouse NIH/3T3 cell line. The 1 and 2 genes are essential for generation of mature B and T lymphocytes.

Expression and hydroxyurea-triggered induction of EGFP upon CRISPR/Cas9-mediated integration into the γ-globin gene of K562 cells.

Objective: To knock-in an EGFP cassette into the γ-globin genes of K562 cells via CRISPR/Cas9, and to assess expression and hydroxyurea (HU)-mediated induction of the targeted EGFP transgene.

Results: The EGFP cassettes were specifically knocked into the Gγ gene. EGFP expression was detected in the targeted cell population and isolated clones.

Mosaicism in CRISPR/Cas9-mediated genome editing.

The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles.

Lentiviral-mediated BCL2 gene knockdown using comparative microRNA adaptive shRNAs.

B-cell lymphoma 2 (BCL2) family proteins play a critical role in tuning cell death processes. Almost in half of all human cancers, a dysregulation in BCL2 family gene expression has been shown which made it an impressive target for human gene therapy as a novel approach in cancers. In this study we will optimize lentiviral-mediated RNA interference (RNAi), recombinant lentiviruses accommodating anti-BCL2 micro adaptive short hairpin RNAs (shRNAs), to downregulate BCL2 in human embryonic kidney 293T (HEK293T) cells to produce stable cell lines.

Effects of Ectoine on Behavior and Candidate Genes Expression in ICV-STZ Rat Model of Sporadic Alzheimer's Disease.

Alzheimer's disease (AD) is pathologically defined by the presence of amyloid plaques and tangles in the brain, therefore, any drug or compound with potential effect on lowering amyloid plaques, could be noticed for AD management especially in the primary phases of the disease. Ectoine constitutes a group of small molecule chaperones (SMCs). SMCs inhibit proteins and other changeable macromolecular structures misfolding from environmental stresses.

Effects of Herbal Compound (IMOD) on Behavior and Expression of Alzheimer's Disease Related Genes in Streptozotocin-Rat Model of Sporadic Alzheimer's Disease.

Sporadic Alzheimer's disease (AD) accounts for over 95% of cases. Possible mechanisms of AD such as inflammation and oxidative stresses in the brain motivate researchers to follow many therapies which would be effective, especially in the early stages of the disease. IMOD, the herbal extract of R.

The Effects of Extract on Expression of Genes in the Streptozotocin-Induced Rat Model of Sporadic Alzheimer's Disease.

Background: Possible mechanisms of Alzheimer Disease (AD) such as inflammation and oxidative stresses in the brain led us to investigate potential AD therapeutics of an herbal extract, with possible role as an anti-inflammatory and anti-oxidant agent. Among different genes which had important role in Sporadic AD (SAD), three genes including and have shown significant statistical diversity in the brains of Alzheimer patients.

Methods: These genes were chosen to be investigated for neuroprotective effects of the extract by comparing the expression level in the hippocampus of Sporadic AD (SAD) rat model using quantitative polymerase chain reaction (qPCR) in the treated and untreated groups.

An enhancer haplotype may influence BCL11A expression levels and the response to hydroxyurea in β-thalassemia patients.

Aim: To identify the BCL11A intron-2 enhancer linkage disequilibrium (LD) block, harboring two previously identified SNPs, associating with the hydroxyurea response in β-thalassemia patients and the functional significance of this region.

Materials & Methods: Several neighboring SNPs were genotyped in our cohort. The associating LD block was identified, and its function studied in K562 erythroid cells via CRISPR/Cas9 genome editing.

Cyclic AMP-Induced p53 Destabilization is Independent of CREB in Pre-B Acute Lymphoblastic Leukemia Cells.

Elevated cAMP levels in B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells attenuate the doxorubicin-induced p53 accumulation and protect cells against apoptosis. cAMP responsive element binding protein (CREB) is a cAMP-stimulated transcription factor that regulates genes whose deregulated expression cooperate in oncogenesis. In the present study, we investigated the role of CREB on inhibitory effect of cAMP on apoptosis and p53 accumulation in BCP-ALL NALM-6 cells.

CREB knockdown inhibits growth and induces apoptosis in human pre-B acute lymphoblastic leukemia cells through inhibition of prosurvival signals.

A majority of acute lymphoblastic leukemia patients overexpress CREB in the bone marrow. However, the functional significance of this up-regulation and the detailed molecular mechanism behind the regulatory effect of CREB on the growth of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) cells has not been elucidated. We demonstrated here that CREB knockdown induced apoptosis and impaired growth of BCP-ALL NALM-6 cells which was associated with caspase activation.

Association Study of IL-4 -590 C/T and DDX39B -22 G/C Polymorphisms with the Risk of Late-Onset Alzheimer's Disease in Iranian Population.

Interleukin-4 (IL-4), an important anti-inflammatory cytokine, is elucidated to regulate amyloid β-induced production of the inflammatory cytokines such as IL-1 and IL-6. It is assumed that IL-4 may involve in the inflammation pathology of surrounding senile plaques in Alzheimer's disease (AD) patients. DEAD (Asp-Glu-Ala-Asp) box polypeptide 39B (DDX39B), appears to be involved in regulation of the inflammatory cytokines which are in correlation with AD pathology.

Mutation screening of the Krüppel-like factor 1 gene using single-strand conformational polymorphism in a cohort of Iranian β-thalassemia patients.

The Krüppel-like factor 1 (KLF1) is an essential erythroid-specific transcription factor. Mutations in the human KLF1 gene have different phenotypic effects, ranging from increased Hb F levels to the disruption of erythropoiesis. Here, we screened 227 Iranian β-thalassemia (β-thal) patients for the presence of KLF1 mutations by using the single-strand conformational polymorphism (SSCP) approach.

Chitosan nanoparticles for siRNA delivery: optimization of processing/formulation parameters.

Chitosan nanoparticles were prepared using ultrasonication methodology at specific amplitudes and times of sonication. Subsequently, small interfering RNA (siRNA) was added to the solution at predetermined values of nitrogen to phosphorous ratio (N/P), and stirring time. Employing response surfaces generated from a statistical model, the effect of sonication time and amplitude, stirring time, and N/P ratio was studied on the particle size, polydispersity, and loading efficiency of prepared siRNA/chitosan nanoparticles.

A defect in the CLIP1 gene (CLIP-170) can cause autosomal recessive intellectual disability.

In the context of a comprehensive research project, investigating novel autosomal recessive intellectual disability (ARID) genes, linkage analysis based on autozygosity mapping helped identify an intellectual disability locus on Chr.12q24, in an Iranian family (LOD score = 3.7).

Detection of HER2 status in breast cancer: comparison of current methods with MLPA and real-time RT-PCR.

Human epidermal growth factor receptor (HER) status is an important prognostic factor in breast cancer. There is no globally accepted method for determining its status, and which method is most precise is still a matter of debate. We here analyzed HER2 mRNA expression by quantitative reverse transcription-PCR (qRT-PCR) and HER2 DNA amplification using multiplex ligation-dependent probe amplification (MLPA).

Optimizing A Lipocomplex-Based Gene Transfer Method into HeLa Cell Line.

One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mam- malian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies.

Utility of the multivariate approach in predicting β-thalassemia intermedia or β-thalassemia major types In Iranian patients.

Recently, five genetic modifiers [β-globin mutations, coinheritance of α-thalassemia (α-thal), XmnI polymorphism and single nucleotide polymorphisms (SNPs) in the BCL11A and HBS1L-MYB loci] were used to predict the β-thal major (β-TM) or β-thal intermedia (β-TI) types in 106 French patients with 83.2% accuracy. The dichotomous grouping was based on the age when the patient received his/her first transfusion (4 years).

Hydroxyurea treatment in β-thalassemia patients: to respond or not to respond?

Hydroxyurea (HU) is a drug that induces fetal hemoglobin production. As a result, HU is widely used to treat β-thalassemia (β-thal) patients. However, the response of these patients to HU varies.

The XmnI and BCL11A single nucleotide polymorphisms may help predict hydroxyurea response in Iranian β-thalassemia patients.

Hydroxyurea (HU), a drug which can reactivate fetal hemoglobin (Hb F) production, is frequently prescribed to β-thalassemia (β-thal) patients. However, transfusion requirements of only a subset of patients are reduced upon HU treatment. Because of its potential side-effects, targeted prescription of HU is imperative.