Real-Time Quantitative PCR to Demonstrate Gene Expression in an Undergraduate Lab

The objective of this teaching note is to develop a laboratory exercise, which allows students to get a hands-on experience of a molecular biology technique to analyze gene expression. The short duration of the biology laboratory for an undergraduate curriculum is the biggest challenge with the development of new labs. An important part of cell biology or molecular biology undergraduate curriculum is to study gene expression. There are many labs to study gene expression in qualitative manner. The commonly used reporter gene expression studies are primarily qualitative. However, there is no hands-on experience exercise to quantitatively determine gene expression. Therefore, it is necessary to design a laboratory exercise that enables the students to carry out cell or molecular biological assays in the desired time. Here we report a laboratory where we can introduce students to gene expression using the real time Quantitative Polymerase Chain Reaction (RT-qPCR) by comparative CT method to analyze expression of genes in Drosophila tissues

Inactivation of Hippo and cJun-N-terminal Kinase (JNK) signaling mitigate FUS mediated neurodegeneration in-vivo

Amyotrophic Lateral Sclerosis (ALS), a late-onset neurodegenerative disorder characterized by the loss of motor neurons in the central nervous system, has no known cure to-date. Disease causing mutations in human Fused in Sarcoma (FUS) leads to aggressive and juvenile onset of ALS. FUS is a well-conserved protein across different species, which plays a crucial role in regulating different aspects of RNA metabolism. Targeted misexpression of FUS in Drosophila model recapitulates several interesting phenotypes relevant to ALS including cytoplasmic mislocalization, defects at the neuromuscular junction and motor dysfunction. We screened for the genetic modifiers of human FUS-mediated neurodegenerative phenotype using molecularly defined deficiencies. We identified hippo (hpo), a component of the evolutionarily conserved Hippo growth regulatory pathway, as a genetic modifier of FUS mediated neurodegeneration. Gain-of-function of hpotriggers cell death whereas its loss-of-function promotes cell proliferation. Downregulation of the Hippo signaling pathway, using mutants of Hippo signaling, exhibit rescue of FUS-mediated neurodegeneration in the Drosophila eye, as evident from reduction in the number of TUNEL positive nuclei as well as rescue of axonal targeting from the retina to the brain. The Hippo pathway activates c-Jun amino-terminal (NH2) Kinase (JNK) mediated cell death. We found that downregulation of JNK signaling is sufficient to rescue FUS-mediated neurodegeneration in the Drosophila eye. Our study elucidates that Hippo signaling and JNK signaling are activated in response to FUS accumulation to induce neurodegeneration. These studies will shed light on the genetic mechanism involved in neurodegeneration observed in ALS and other associated disorders

Total RNA Extraction from Transgenic Flies Misexpressing Foreign Genes to Perform Next Generation RNA Sequencing

Due to absence of transgenic approaches in Notopthalmus Viridescens (newt), and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress unique genes from newt. Novel newt genes cloned, and inserted at attP site in Drosophila were misexpressed ubiquitously using tubulin Gal-4. Sample (total RNA) for RNA sequencing was collected at 3rd instar larval stage during which major developmental events takes place in Drosophila. Total RNA was extracted, and purified using RNA clean and ConcentratorTM. RNA quality was quantitated by calculating absorbance at 260 nm (A260) and 280 nm (A280) wavelengths using Nanodrop 2000 spectrophotometer. Good quality samples had A260/ A280 ratio greater than 2 and a peak at 260 nm. Our results show that following this protocol high quality of RNA was obtained. These high quality RNA samples were used for downstream processes e.g. Next generation RNA sequencing. Of the total 36,099 transcripts in Drosophila, 34,967 transcripts were detected, and 2775 transcripts were significantly regulated by misexpressing foreign gene (Unique gene from newt) in Drosophila . Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. Wingless/Wnt was one of the important evolutionarily conserved pathway that was differentially regulated

Comparative Transcriptomic Analysis and Structure Prediction of Novel Newt Proteins

Notophthalmus viridescens (Red-spotted Newt) possess amazing capabilities to regenerate their organs and other tissues. Previously, using a de novo assembly of the newt transcriptome combined with proteomic validation, our group identified a novel family of five protein members expressed in adult tissues during regeneration in Notophthalmus viridescens. The presence of a putative signal peptide suggests that all these proteins are secretory in nature. Here we employed iterative threading assembly refinement (I-TASSER) server to generate three-dimensional structure of these novel Newt proteins and predicted their function. Our data suggests that these proteins could act as ion transporters, and be involved in redox reaction(s). Due to absence of transgenic approaches in N. viridescens, and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress these genes. Expression of 2775 transcripts were compared between these five newly identified Newt genes. We found that genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. To validate the RNA Seq. data, expression of six highly regulated genes were verified using real time Quantitative Polymerase Chain Reaction (RT-qPCR). These graded gene expression patterns provide insight into the function of novel protein family identified in Newt, and layout a map for future studies in the field

Insights into Regeneration Tool Box: An Animal Approach

For ages, regeneration has intrigued countless biologists, clinicians, and biomedical engineers. In recent years, significant progress made in identification and characterization of a regeneration tool kit has helped the scientific community to understand the mechanism(s) involved in regeneration across animal kingdom. These mechanistic insights revealed that evolutionarily conserved pathways like Wnt, Notch, Hedgehog, BMP, and JAK/STAT are involved in regeneration. Furthermore, advancement in high throughput screening approaches like transcriptomic analysis followed by proteomic validations have discovered many novel genes, and regeneration specific enhancers that are specific to highly regenerative species like Hydra, Planaria, Newts, and Zebrafish. Since genetic machinery is highly conserved across the animal kingdom, it is possible to engineer these genes and regeneration specific enhancers in species with limited regeneration properties like Drosophila, and mammals. Since these models are highly versatile and genetically tractable, cross-species comparative studies can generate mechanistic insights in regeneration for animals with long gestation periods e.g. Newts. In addition, it will allow extrapolation of regenerative capabilities from highly regenerative species to animals with low regeneration potential, e.g. mammals. In future, these studies, along with advancement in tissue engineering applications, can have strong implications in the field of regenerative medicine and stem cell biology

Regeneration Data: RNA Sequences

Abstract for paper for which this research was collected: Notophthalmus viridescens (Red-spotted Newt) possess amazing capabilities to regenerate their organs and other tissues. Previously, using a de novo assembly of the newt transcriptome combined with proteomic validation, we identified a novel family of 5 protein members expressed in adult tissues during regeneration in Notophthalmus viridescens. Due to absence of transgenic approaches in the Notophthalmus, and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress these genes. Samples for RNA sequencing were collected at third instar larval (L3) developmental stage. Out of the total 36,099 transcript models in Drosophila, 34,967 transcripts were detected. The expression of more than 27,000 transcripts was compared between these 5 newly identified Newt genes. C1 regulated 2220 transcripts; C2 regulated 1383 transcripts, C3 regulated 1446 transcripts, C4 regulated 182 transcripts, and C5 regulated 2212 transcripts. Very few regulated genes were found to be specific to particular Newt protein. Genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched

Viropana (vna) 4

Viropana (vna) 4 ID – vna 4 Details – Gene from a novel protein family belonging to the regeneration toolkit of Notophthalmus viridescens Base pairs – 50

Viropana (vna) 3

Viropana (vna) 3 ID – vna 3 Details – Gene from a novel protein family belonging to the regeneration toolkit of Notophthalmus viridescens Base pairs – 42

Viropana (vna) 2

Viropana (vna) 2 ID – vna 2 Details – Gene from a novel protein family belonging to the regeneration tool kit of Notophthalmus viridescens Base pairs – 39