This document discusses different types of reporter genes that are used in plant functional genomics studies. It describes scorable reporter genes like green fluorescent protein (GFP), yellow fluorescent protein (YFP), and β-glucuronidase (GUS) which produce quantifiable phenotypes through enzyme assays. It also describes selectable reporter genes like antibiotic and herbicide resistance genes which allow for selection of transformed cells. Reporter genes are useful for identifying gene expression patterns, performing gene expression assays by fusing the reporter to a gene of interest, and assessing transformation/transfection efficiency. The document provides examples of using GFP fused to the XPR1 gene to study its subcellular localization in tobacco cells.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
Genetic manipulation of plant and animal cells have to be confirmed for further application. One such confirmatory method is the use of stains/dyes which produces fluorescence when the recombination is successful.
Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com
What is Genome,Genome mapping,types of Genome mapping,linkage or genetic mapping,Physical mapping,Somatic cell hybridization
Radiation hybridization ,Fish( =fluorescence in - situ hybridization),Types of probes for FISH,applications,Molecular markers,Rflp(= Restriction fragment length polymorphism),RFLPs may have the following Applications;Advantages of rflp,disAdvantages of rflp, Rapd(=Random amplification of polymorphic DNA),Process of rapd, Difference between rflp &rapd
In nuclear biology and molecular biology, a marker gene is a gene used to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Genomic library and shotgun sequencing. It includes the topics about genomic library,construction method, its uses and applications, shotgun sequencing, difference between random and whole genome sequencing, its advantages and disadvantages etc.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Separation is brought about through molecular sieving technique, based on the molecular size of the substances. Gel material acts as a "molecular sieve”.
Gel is a colloid in a solid form (99% is water).
It is important that the support media is electrically neutral.
Different types of gels which can be used are; Agar and Agarose gel, Starch, Sephadex, Polyacrylamide gels.
this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers
Gene trapping is a type of insertional mutagenesis that disrupts gene function by the integration of a vector in the intergeneric sequences. It provides an important and unique method for studying the relationship between gene expression and function and to characterize novel genes and analyze their importance in biological phenomena. It is insertional based gene discovery that utilizes random integration of reporter gene construct into genome and produce dominant expression phenotype.
It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion. The transformed cells are selected on the basis of selectable markers. Insertion events are detected and the trapped lines are established. This technique has been used to identify tissue specific and temporally regulated genes in plants and mice. It proves to be a powerful tool of functional genomics.
thyroid carcinoma with specific empahsis on papillary thyroid carcinoma, history, risk factors, involved signalling pathway of MAPK, world wide pathogenesis status, its proposed targetting, involvmetn in cancer types
An oncogene is a gene that has the potential to cause cancer. In tumor cells, they are mutated or expressed at high levels. Most normal cells undergo a programmed form of rapid cell death (apoptosis) when critical functions are altered.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
2. Reporter genes
• Gene whose products are easily detected or
monitered
• A gene that is used to `tag' another gene or
DNA sequence of interest
Identifying whether a certain gene has been
taken up by cell
Measurement of gene expression
3. • Easily quantifiable
• Relatively rapid degradation of the enzyme
• Lack of endogenous activity in the concerned cell
• Should not be toxic to cells
• Assay should be sensitive and reliable
4.
5. Types of Reporter gene
Scorable reporter genes
• Expression of this results in quantifiable
phenotype
• Easily detected through highly sensitive enzyme
assays
Selectable reporter genes
• Expression of resistance to a toxin
• Selection of tranformants from nontransformants in
growth media containing selective agent
6. Scorable reporter gene
Green Fluorescent Protein
• Derived from jellyfish Aequorea victoria
• Formed by nucleophilic reaction between C-ter of S with N-ter of G,
formed imidazoline heterocyclic ring which oxidise with Y to yield
floroscence
Variants of GFP
Yellow Fluorescent Protein
• Formed by mutation of Thr 203 residue to tyrosine
Blue Fluorescent Protein
• Modification of tyr66 to his
Cyan Fluorescent Protein
Modification of tyr66 to tryptophan
• Derived from Discosoma striata (Ds Red)
• Alternation of >30 amino acid to yield RFP-1
Shaner N.C et al, Nature Biotech 22,1562-1572 (2004)
7. Fruit series
Cubitt, A., R. Heim, S. Adams, A.
Boyd, L. Gross, R. and R. Tsien
(1995) Understanding, improving
and Using Green Fluorescent
Proteins. Trends in Biochemical
Sciences, 20:448-455.
9. GUS
• Derived from E.Coli
• uid A gene code for 12- -glucuronidase enzyme
• Enzymatic cleavage of X-Gluc (5-bromo-4-chloro-3-indolyl -D-
glucuronide) undergoes an oxidative dimerization to yield an indigo blue
precipitate
Luciferase
• Bacterial luciferase : Vibrio harveyi (luxA/luxB genes)
• Firefly (Photinus pyralis) luciferase (luc gene)
β-galactosidase (LacZ)
• Derived from:E.coli
• catalyzes the hydrolysis of X-Gal producing a blue precipitate
10. Selectable reporter gene
Antibiotic Resistance Genes
Neomycin phosphotransferase II (npt II gene)
• Derived from the transposon Tn5 code foraminoglycoside 3`
phosphotransferase
• Resistance to the antibiotic kanamycin neomycin by
phosphorylation
Hygromycin phosphotransferase (hpt gene)
• Derives from E. coli
• Resistant against hygromycin by phosphorylation
11. Herbicide Resistance Markers
Phosphinothricin acetytransferase (pat/bar gene)
• Derived from Streptomyces hygroscopicus
• Converts herbicides into acetylated forms
Resistant against Bialophos, phosphinothricin and glufosinate
Enolpyruvylshikimate phosphate synthase (epsps/aroA genes)
• Derived from Agrobacterium sp CP4
• Resistance against glyphosate which blocks the activity of EPSP synthase,
a key enzyme involved in the biosynthesis of aromatic amino acid
Bromoxynil nitrilase (bxn gene)
• Derived from Klebsiella pneumoniae
• The herbicide bromoxynil inhibits photosynthesis (photosystem II)
• Encode a specific nitrilase that converts bromoxynil to its primary
metabolite 3,5-dibromo-4-hydroxybenzoic acid
12. Reporter gene for functional genomics
Identify a promoter, to study the expression pattern and strength
of the promoter
• Reporter gene is simply placed under the control of the target
promoter
Gene expression assays
• Reporter is directly attached to the gene of interest to create
a gene fusion
• The two genes are under the same promoter elements and are
transcribed and then translated into protein
Transformation and transfection assays
• Reporter genes expressed under their own promoter independent
from that of the introduced gene of interest
• Reporter gene can be expressed constitutively or inducibly
13. T. Weber, R. Köster / Methods 62 (2013) 279–
291
16. Schematic diagrams of the PtMCP promoter::GUS and CaMV35S promoter::GUS construct. RB, right
border; LB, left border; NOSpro, nopaline synthase promoter; NPT-II, neomycin
phosphotransferase (II) coding region; NOS-T, nopaline synthase terminator; GUS,
β-glucuronidase gene; PtMCPpro, PtMCP promoter; CaMV35Spro, cauliflower mosaic
virus (CaMV) 35S promoter. The insertion position of the PtMCP promoter in the vector is
indicated by the restriction enzyme sites SacI and KpnI
17. (C and G), positive
controls (CaMV35S
promoter::GUS)
(D and H), negative
controls (wild-type
plants
18.
19.
20. • XPR1 as Pi exporter
• Homolog with arabidopsis PHO1
• Has 2 domain SPX and EXS
• SPX Domain is not required for Pi export
• Mostly localised in Golgi and trans golgi network using GFP as
marker
XPR1
TMX
2X CaMV SPX 4TM EXS GFP
2XCaMV 4MT EXS GFP
21. Co-expression of XPR1-GFP with different
subcellular markers in tobacco epidermal
cells.
Tobacco leaves were infiltrated with A.
tumefaciens strains harboring free GFP as
a control
22. Co-expression of XPR1-GFP
with different subcellular
markers in tobacco
epidermal cells.
Tobacco leaves were
infiltrated with A.
tumefaciens strains
harboring free GFP as a
control
