RNA interference (RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner. RNAi has been observed in all eukaryotes, from yeast to mammals. The RNAi pathway is thought to be an ancient mechanism for protecting the host and its genome against viruses and rogue genetic elements that use double-stranded RNA (dsRNA) in their life cycles. They have also been shown to play a role not only in mRNA and dsRNA stability/degradation, but also in regulation of translation, transcription, chromatin structure, and genome integrity. In plants and animals, RNA silencing has been adapted to play a critical role in regulation of cell growth and differentiation using a class of small RNAs. In the RNA interference process, the dsRNAs get processed into 20-25 nucleotide (nt) small RNAs by an RNase III-like enzyme called Dicer. Then, the small RNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The small RNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand. The small RNAs that provide target specificity to the silencing machinery includes short interfering RNAs (siRNAs), repeat-associated siRNAs (rasiRNAs), and microRNAs (miRNAs) and is distinguished by their origin. siRNAs are processed from dsRNA precursors made up of two distinct strands of perfectly base-paired RNA, while miRNAs originate from a single, long transcript that forms imperfectly base-paired hairpin structures. siRNAs were originally identified as intermediates in the RNAi pathway after induction by exogenous dsRNA; however, endogenous sources of siRNAs have now been recognized. The endogenous siRNAs are derived from repetitive sequences within the genome, and are termed repeat-associated siRNAs, or rasiRNAs. miRNAs were discovered through their critical roles in development and cellular regulation, and represent a large class of evolutionarily conserved RNAs. miRNAs have always been recognized as being of endogenous origin. RNA interference has emerged as a natural mechanism for silencing gene expression over the past decade. This ancient cellular antiviral response can be harnessed to allow specific inhibition of the function of any chosen target genes, including those involved in causing diseases such as cancer, AIDS, and hepatitis. It is already proving to be an invaluable research tool, allowing much more rapid characterization of the function of known genes. More importantly, the technology considerably bolsters functional genomics to aid in the identification of novel genes involved in disease processes. Last but not the least the technology can be harnessed as a novel therapeutic agent and is suitable for combating viral diseases, cancers and inflammatory diseases.

Imgenex (San Diego) recently launched the pSuppressorAdeno construction kit for adenovirus mediated gene knockdown. The kit provides the ability to infect a broad range of cell types, including many primary cell lines as well as dividing and nondividing cells, according to a company official. The kit also offers the flexibility to validate sequences using the nonviral expression plasmid prior to construction of adenoviruses, notes Sujay K. Singh, Ph.D., president and CEO of Imgenex, which markets plasmid-based RNA interference (RNAi) products. “One of the greatest advantages is the ability of recombinant adenovirus vectors to reduce gene expression both in vitro and in vivo,” he adds. RNAi, initially considered a bizarre attribute of petunias and later a gene-silencing mechanism in worms, is creating a stir as one of the hottest new technologies in molecular biology. It is revolutionizing the field of functional genomics.

IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of  IMGENEX Corporation San Diego, USA.  Find out more information about   RNA interference.

All the cellular processes in living cells such as growth, development, morphogenesis and cellular differentiation are a product of gene expression programs involving complicated transcriptional regulation of several genes. This process of transcriptional regulation is tightly controlled and coordinated by proteins called transcriptional regulators. These transcriptional regulators and factors are DNA-binding proteins that bind to the promoter or enhancer sequences on the DNA and facilitate either transcriptional repression or activation.

There are three principal types of transcription factors. These include basal transcription factors, upstream transcription factors and inducible transcription factors. The basic structure of every transcriptional factor mainly contains a DNA-binding domain and an activator domain. DNA-binding motifs found in transcription factors include zinc-finger, helix-loop-helix, helix-turn-helix, leucine zipper and high-mobility groups, based on which transcription factors are classified. The activator domain of these transcription factors interacts with components of transcription machinery such as RNA polymerases and associated transcription regulators.

Regulation of transcriptional factors is a complex mechanism that ensures exact spatio-temporal expression of genes. In response to a specific cellular stimulus, these trans-regulatory factors are activated in a sequential manner. Upon activation, these factors recruit transcriptional co-regulators such as histones that function as co-activators or co- repressors and aid in modifying chromatin structure. Altered activation of these regulators is often associated with various pathologies such as chronic disorders and malignancies. Recent studies are concentrating on developing improved disease treatment strategies through identification of different transcription factor-binding patterns and blocking them.

There are several families of trans-regulatory factors that control critical cellular signaling cascades involved in cell proliferation, survival, lineage development and cellular differentiation. These include Rel/NF-kB family, AP-1 family, STAT family of transcription factors, homeodomain proteins, DNA-binding proteins, POU transcription factors, nuclear hormone receptor family, p53 family and E2F family.

IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of IMGENEX Corporation, San Diego, USA.Find out more information about   Transcriptional Factors.

Early development and differentiation of nascent T cells that migrate from bone marrow to become mature, naïve T cells, which are capable of responding to antigen takes place inside the thymus. Around 10¹⁰ TCR (T cell receptor) variations are generated in developing T lymphocyte clones through a random process of somatic cell gene reorganization. During this process, often T-cells recognizing self-antigens are generated. Due to the ability of these self-reactive T-cells to elicit an autoimmune attack, they are permanently removed by the thymus through negative selection and clonal deletion. But, some of them manage to escape the thymic defenses and harbor themselves in the peripheral lymphoid organs. In periphery, T lymphocytes undergo further differentiation into effectors of various immune functions.

One of many immunotolerance mechanisms that immune system has developed to distinguish between self and non-self antigens is regulatory T cells or Tregs. These cells are recently characterized specialized T-cell subsets that actively suppress a variety of immune responses. Researchers have broadly classified Tregs into natural and adaptive Tregs. Natural Tregs are CD4⁺CD25⁺ T-cells that originate in the thymus and play a significant role in immune homeostasis and protection against autoimmunity. Adaptive Tregs are non-regulatory CD4⁺ T-cells that have up-regulated CD25 expression during pathological and inflammatory conditions such as cancers and infections.

Although the principal immunosuppressive mechanism of Tregs remains elusive, several in vivo experimental models have indicated that Tregs secrete large amounts of immunosuppressants including IL-9, IL-10 and TGF-β upon activation. These lymphokines are capable of inhibiting activation of Th1, Th2 cells and CTLs required for cell-mediated immunity, inflammation and antibody production. Certain recent experimental data and results even indicate that IL-2-IL-2R signaling is vital for development, maintenance, survival, expansion and suppressive activity of Tregs. Increased expression of certain other characteristic markers including CTLA-4, glucocorticoid-inducible tumor necrosis factor receptor (GITR) and OX40 has been identified on Tregs whose function inside these cells is still not clear. The TCRs displayed on Tregs are capable of recognizing and interacting with any peptide-MHC class II ligand having certain range of avidity. But, the contribution of TCR signaling and role of TCR-ligand interactions towards regulatory T-cell development needs to be determined.

Several elegant experiment using transgenic mice and retrovirus mediate over expression studies, researchers have identified FoxP3, a transcription factor, to be a specific molecular marker essential for the development and function of Tregs. The primary evidence regarding the involvement of FoxP3 in the development of Tregs was provided by the experiments of Sakaguchi et al, (ref ?) in patients suffering from IPEX, a rare and fatal human autoimmune disorder. In these patients, mutated FoxP3 gene causes improper development of Tregs resulting in hyperactivation of T-cells reactive to self-antigens. Recently, experiments have clearly shown that retroviral mediated introduction of FoxP3 into conventional CD4+ T-cells converts them into regulatory T-cells.

The emergence of regulatory T-cells and role of FoxP3 as a critical player in the negative control of a of various normal and pathological immune responses holds great promise for the development of novel therapies useful for the treatment of autoimmune diseases in humans. However, there are several questions that remain to be answered including the basic biology of the Tregs, various ligands responsible for thymic selection of these cells, the exact function of FoxP3 in relation with various markers present on Tregs and most importantly, the mechanisms by which Tregs exert their suppressive effects. A better understanding of manipulating FoxP3 and Tregs would enable us to harness the tremendous therapeutic potential in various clinical situations including Type I diabetes, Multiple sclerosis, GVHD, rheumatoid arthritis, allergy, and cancers.

IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of IMGENEX Corporation, San Diego, USA.  Find out more information about Regulator T-cells(Treg).

Immunology is a broad branch of biomedical science that covers the study of all aspects of the immune system in all organisms. The immune system is the body's defense against infectious organisms and other invaders. Through a series of steps called the immune response, the immune system attacks organisms and substances that invade our systems and cause disease. The immune system is made up of a network of cells, tissues, and organs that work together to protect the body. The immune system is the body’s natural defence in combating organisms. The immune system usually have two lines of defence: the innate immune system representing a non-specific (no memory) response to antigen (substance to which the body regards as foreign or potentially harmful) and the adaptive immune system, which displays a high degree of memory and specificity. The innate system represents the first line of defence to an intruding pathogen and includes various cells like the natural killer (NK) cells, mast cells dendritic cells and phagocytes. Besides there are molecules like complement, acute phase proteins (APP) and interferons (IFNs) which work in concert with the cells of the innate immune system and which foster close functional links with their adaptive counterpart. The adaptive immune system is further divided into humoral and cellular components.  Cell-mediated immunity, also known as delayed-type hypersensitivity (DTH) or Type IV Hypersensitivity, is an immune response that does not involve antibodies but rather involves the activation of macrophages, natural killer cells (NK), antigen-specific cytotoxic T-lymphocytes, and the release of various cytokines in response to an antigen. The humoral immune response (HIR) is the aspect of immunity that is mediated by secreted antibodies, produced in the cells of the B lymphocyte lineage (B cell). When activated by foreign antigen, B cells undergo proliferation and mature into antibody secreting plasma cells which posses the ability to secrete soluble proteins (antibodies). Antibodies which are classified into five different types (known as isotypes), namely IgM, IgG, IgA, IgE and IgD, have two roles to play - the first is to bind antigen and the second is to interact with host tissues and effector systems in order to ensure removal of the antigen. Thus the immune system generally is protective, however the same immunologic mechanisms that defend the host at times may result in severe damage to tissues and, occasionally, may cause death.

Conceptualizing the natural antigen- antibody development & interaction, Imgenex Corp. develops and commercializes novel reagents for the scientific study of human biology and disease and for the production of new diagnostic assays and potential therapies of such diseases. These novel reagents include antibodies, gene and protein expression systems, and arrays of various cells and tissues for use in studies of functional genomics. Areas of biological interest at IMGENEX include cancer, apoptosis (programmed cell death), molecular signaling pathways, cellular aging, and metabolic and infectious diseases.

IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of IMGENEX Corporation, San Diego, USA. Find out more information about immunology.

FOXP3 is a master regulator of immune homeostasis expressed specifically in CD4⁺ CD25⁺ T regulatory cells controlling their growth, development and function.  FOXP3 significance in the normal development of Tregs is better elucidated with the fact that mutated FOXP3 results in a rare and fatal early onset autoimmune disorder in humans called XLAAD/IPEX (human immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome), a condition phenotypically similar to Scurfy in mice. 

FOXP3 is primarily an oligomeric, transcriptional repressor protein that belongs to the P subfamily of forkhead (FKH)-winged helix family of transcriptional factors. Members of this subfamily are forkhead (FKH) box proteins characterized by the presence of a highly conserved C-terminal winged-helix/FKH DNA binding domain (DBD) and centrally-located C2H2 zinc finger domain and leucine zipper domain.  Apart from these, an additional N-terminal proline rich region is present in FOXP3, whose function is yet to be understood. Studies have shown that FOXP3 is a nuclear-localized protein that specifically trans-represses NF-AT-induced expression of cytokines and other transcriptional factors in Tregs including IL-2, IL-4, IFN-gamma and NF-κB.

However, FOXP3 is not the sole master switch regulating the origin and development of CD4⁺ CD25⁺ Tregs. Studies have confirmed the existence of splice variant forms FOXP3 that are specifically expressed in humans but are lacking in mouse. Cloning and RT-PCR analysis from mRNA of CD4⁺ CD25⁺ T regulatory cells (Allan et. al, Smith et. al) has shown that these cells express two different alternatively spliced variant forms of FOXP3. While the FOXP3Δ2 variant had a deleted 105bp exon2 region, there was another FOXP3Δ2, Δ7 variant that had an additional 81bp exon7 deletion apart from exon2 deletion. The predicted molecular weight of this FOXP3Δ2 is ~4KDa lower than the molecular weight of FOXP3.  Transient transfection assays using Jurkat cells suggest that the FOXP3Δ2 is novel splice variant that functions as a transcriptional repressor protein and acts in cohort with FOXP3 causing a significant suppression of cytokines and up-regulating the expression of various Treg-associated markers.

The existence of the splice variant forms of FOXP3 protein suggests an additional level of complexity related to the biology of FOXP3.  A lot research needs to be done so as to elucidate the physiological and functional importance of FOXP3 splice variant forms towards maintaining immune homeostasis in Tregs and preventing autoimmune disorders.

Continuing its efforts towards supporting research on FOXP3, IMGENEX has come up with an antibody that specifically recognizes the FOXP3Δ2 splice variant.

IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of IMGENEX Corporation, San Diego, USA.  Find out more information about FOXP3.

As a major target for dopamine-activated adenylyl cyclase and protein kinase A in the striatum, DARPP-32 plays a central role in regulating the efficacy of dopaminergic neurotransmission and can act either as a phosphatase or kinase inhibitor in a contextually dependent manner. Growing evidence points to DARPP-32 as a key mediator/modulator in numerous signal transduction cascades implicated in a growing number of neurological disorders such as schizophrenia and depression, as well as in drug abuse and addiction.  For example, it has been shown that acute exposure of mice to cocaine increases phosphorylation of DARPP-32 at Thr34 with a concommitment decrease in phosphorylation at Thr75, whereas chronic exposure leads to a reversal of this phospho profile.  It has also been observed that many schizophrenic patients show decreased levels of DARPP-32 in the prefrontal cortex.

While the precise role that DARPP-32 plays in these psychiatric disorders is still being elucidated, the existing body of literature clearly portrays DARPP-32 as playing a central role in multiple signaling pathways.  Further research will determine neuronal subpopulations where phosphorylation of DARPP-32 occurs as well as the functional importance of previously identified phospho sites of DARPP-32.

IMGENEX offers over one-thousand Neuroscience-related antibodies such as recently cited anti-DARPP-32 (pan and phosphospecific), Dopamine D1 and D2 Receptors (IHC validated), 5-HT Receptors (such as 5-HT1B), Adenosine Receptor, NMDA and more!

IMGENEX India Pvt Ltd. the only biotech company in Orissa and one of its kinds in Eastern India. IMGENEX India started in Oct as an outsourcing branch of IMGENEX Corporation, San Diego, USA. Find out more information about DARPP-32.