Endocytosis of GABA receptor: Signaling in nervous system.

GABA (ᵞ-aminobutyric acid), is the principal neurotransmitter known for its inhibitory role in chemical synapses. Being localized primarily in the central nervous system (CNS) it maintains a balance between excitatory (regulated by another neurotransmitter, glutamate) and inhibitory impulses. GABA acts by binding to their specific receptors GABAA and GABAB when released into the post-synaptic nerve terminal. Both of these receptors are responsible for fast and slow inhibition of neurotransmission, respectively. GABAA is a ligand-gated ionopore receptor which opens the Cl- ion channel and decreases the resting potential of the membrane resulting into inhibition of the synapse. On the other hand, GABAB is a metabotropic receptor which increases the K+ ion levels preventing Ca+ ion release inhibiting the release of other neurotransmitters into the presynaptic membrane. The internalization and trafficking of these receptors is also conducted through distinct pathways and mechanism, discussed in detail in the chapter. Without the desired levels of GABA in the body, the psychological and neurological states of brain get hard to maintain. Various neurodegenerative diseases/disorders have been associated to low levels of GABA, such as anxiety, mood disorders, fear, schizophrenia, hungtington's chorea, seizures, epilepsy, etc. The allosteric sites present on GABA receptors have been proved to be potent drug targets to pacify the pathological states of these brain related disorders to an extent. Further in depth studies focussing on the subtypes of GABA receptors and their comprehensive mechanism are required to explore new drug targets and therapeutic avenues for effectual management of GABA related neurological diseases.

CRISPR-based genome editing of zebrafish.

CRISPR/Cas9, once discovered as an adaptive immune system in bacteria, has emerged as a disruptive technology in the field of genetic engineering. Technological advancements in the recent past has enhanced the applicability of CRISPR/Cas9 tool for gene editing, gene therapies, developmental studies and mutational analysis in various model organisms. Zebrafish, one of the excellent animal models, is preferred for conducting CRISPR/Cas9 studies to assess the functional implication of specific genes of interest. CRISPR/Cas9 mediated gene editing techniques, such as, knock-out and knock-in approaches, provide evidences to identify the role of different genes through loss-of-function studies. Also, CRISPR/Cas9 has been proved to be an efficient tool for designing disease models for gene expression studies based on phenotypic screening. The present chapter provides an overview of CRISPR/Cas9 mechanism, different strategies for DNA modifications and gene function analysis, highlighting the translational applications for future prospects, such as screening of drug toxicity and efficacy.

Design of non-viral vector with improved regulatory features towards therapeutic application.

Viral vectors based gene therapy is often compromised by adverse immunological reactions raising safety concerns. Hence, improved design and development of non-viral vectors with strong regulatory regions is desired. We describe the design of a non-viral mammalian expression vector in which the primary transgene (a truncated dystrophin gene linked with Duchenne muscular dystrophy (DMD)) named microdystrophin delR4-R23/delCT (MD1) is under the transcriptional control of elements of desmin locus control region (DES-LCR). The designed vector, named as DES-LCR/MD1-EGFP, was constructed by cloning two fragments into the pBluescript backbone. Fragment 1 contains DES-LCR enhancer and DES-LCR promoter region while fragment 2 contains MD1 transgene and reporter EGFP (enhanced green fluorescent protein) gene separated by linker P2A (2A peptide). This vector design provides a framework for strong regulation with non-viral features. This design forms the foundation for application in conditions linked to multisystem diseases.

A Systematic Bioinformatics Approach to Motif-Based Analysis of Human Locus Control Regions.

Locus control regions (LCRs), cis-acting, noncoding regulatory elements with strong transcription-enhancing activity, are conserved in sequence and organization, and exhibit strict gene-specific expression. LCRs have been reported and studied in several mammalian gene systems, signifying that they play an important role in eukaryotic gene expression control. Their highly regulated, stable, and precise levels of expression have made them a strong candidate for use in gene therapy vectors. In this study, we attempted to determine the unique signatures of human LCRs by analyzing a data set of LCR sequences for the presence of motifs through systematic bioinformatics approach. Using web-based regulatory sequence analysis tools (RSAT), motif-based analysis was performed. Detected significant motifs were analyzed further for their identity using Tomtom tool. RSAT analysis revealed that significant motifs are existent within the LCRs. Identity analysis using Tomtom showed that detected significant motifs were comparable with known transcription factor (TF) binding sites and the top scoring motifs belong to zinc finger-containing proteins, an important group of proteins involved in a variety of cellular activities. Correspondence to segment of known motif indicates the biological relevance of the detected motifs. Motif-based analysis is valuable for analyzing the various characteristics of sequences, notably TF binding models in this study. Owning to their unique expression control abilities, LCRs form an important component of integrating vectors, therefore identification of unique signatures present within LCR sequences will be instrumental in the design of new generation of regulatory elements containing LCR sequences.