This document discusses the use of fluorescent proteins in current biological research. It begins with an overview of the development of optical microscopy and fluorescence techniques. It then focuses on the green fluorescent protein (GFP) and how it has been used as a molecular tag to study protein expression and interactions in living cells through techniques like gene delivery, transfection, viral infection, FRET, and optogenetics. The document concludes that fluorescent proteins have revolutionized cell biology by enabling the real-time visualization and control of molecular pathways and signaling processes in living systems.
Proteomics, definatio , general concept, signficanceKAUSHAL SAHU
INTRODUCTION
GENERAL CONCEPT
WHY PROTEIOMIC NECESERY?
WHAT PROTEOMIC CAN ANSWER?
PRTEOMICS- ANALYSIS AND IDENTIFICATION OF PROTEIN
TWO-DIMENSIONAL SDS-PAGE
MASS SPECTROMETERS
SIGNIFICANCE OF STUDY AN ITS IMPORTANCE
APPLICATIONS
CHALLENGES
CONCLUSIONS
REFERENCES
Proteomics, definatio , general concept, signficanceKAUSHAL SAHU
INTRODUCTION
GENERAL CONCEPT
WHY PROTEIOMIC NECESERY?
WHAT PROTEOMIC CAN ANSWER?
PRTEOMICS- ANALYSIS AND IDENTIFICATION OF PROTEIN
TWO-DIMENSIONAL SDS-PAGE
MASS SPECTROMETERS
SIGNIFICANCE OF STUDY AN ITS IMPORTANCE
APPLICATIONS
CHALLENGES
CONCLUSIONS
REFERENCES
A Beginner’s Guide to the Principles and Applications of FRETExpedeon
FRET, or Fluorescence Resonance Energy Transfer, was first described more than 50 years ago. The availability of new dyes and detection technology has resulted in a much wider use of the application in recent years, especially in biomedical R&D.
Biotechnophysics: DNA Nanopore SequencingMelanie Swan
Biophysics (not merely bioengineering) is required to understand the fundamental mechanisms of biology in order to make technologies (bench and bioinformatic) for understanding them
A Beginner’s Guide to the Principles and Applications of FRETExpedeon
FRET, or Fluorescence Resonance Energy Transfer, was first described more than 50 years ago. The availability of new dyes and detection technology has resulted in a much wider use of the application in recent years, especially in biomedical R&D.
Biotechnophysics: DNA Nanopore SequencingMelanie Swan
Biophysics (not merely bioengineering) is required to understand the fundamental mechanisms of biology in order to make technologies (bench and bioinformatic) for understanding them
Present day analytical method such as gas chromatography- mass spectrophotometry (GC-MS), liquid chromatography (LC-MS) and atomic absorption chromatography (AAS) are straight forward approach with high sensitivity, selectivity, accuracy and reproducibility. These are succeeded in selective detection and identification of harmful contaminants from environmental, tissues or food samples. Mean while, suffers from a number of drawbacks such as, they are limited to a pre-determined set of substances, restricted to pre-programmed scope of analytes, fails to indicate bioavailable concentration, time consuming, expensive and requires lot of expertise. Bacteria have long been served as model for explaining the dose response dependent toxicity for specific chemicals in monitoring of environmental contamination. Ever since the conception of bacterial bioreporter in environmental microbiology has been an increases interest in the construction of well challenged report system based on genetic engineering concept. Bacterial bioreporter are living microorganisms that responds to changes in the environment by displaying specific and easily measurable signal. Based on gene expression in presence of toxic/ stress, resistance to heavy metal/ antibiotics, metabolism of organic compounds and other chemicals are explored for construction of reporter system in bacteria by fusion of specific reporter gene with promoter for detection of harmful contaminants. Assaying by using bioreporter for more complex real sample is more challenging because of presence of inhibitory compounds, unknown compounding effects on behavior and sorptive effects of matrix. The bacterial reporters are also explored for foodstuffs for monitoring of arsenic and tetracycline in rice and milk respectively. There are clear, assay miniaturization may provide the basis for the future incorporation of reporter cells into small devices, synthetic biology efforts will further streamline the construction and engineering of the new reporter strains. There are regulatory issues limiting the application of bioreporter assays, owing to the fact that the bacterial in question are genetically modified.
The final presentation for BTC1700H for team Wild Type. The presentation includes financial and clinical data regarding expression and purification of GFP from an unknown sample of DNA.
Making organelles visible - in planta and in societasAnne Osterrieder
This is the presentation I gave in Salzburg at the Annual Meeting of the Society for Experimental Biology, July 2012, for receiving the President's Medal for Education and Public Affairs.
http://www.sebiology.org/meetings/Salzburg2012/pres_meds.html
Transfection is the process of introduction of foreign DNA into the nucleus of eukaryotic cell. The cells which has incorporated exogenous DNA are called transfectants.
There are two types of Transfection possible,
Transient and
Stable Transfection.
In transient Transfection, the foreign DNA will not get incorporated in to the host genome, but genes are expressed for limited period of time (24-96 hrs).
Stable transfectants will have the foreign DNA incorporated into the genome.
Southern, Northern and Western Blotting methods in genetic EngineeringRavi Raj
Blotting is a method in which a macromolecule is immobilized on a blotting matrix and subsequently probed with a detectable ligand to determine whether the macromolecule binds that specific ligand. The immobilized macromolecule can be DNA, RNA or protein, in which case one generates DNA blots (Southern blots), RNA blots (Northern blots) (1), or protein blots (Western blots).
Using the 3D Cell Explorer-fluo for fluorescence and holotomographic imagingMathieuFRECHIN
Nanolive’s 3D Cell Explorer allows for the creation of very powerful 3D images and 4D time lapses
of living cells with very high spatio-temporal resolution (x,y:180nm; z:400nm; t:1.7sec). Moreover,
imaging with the 3D Cell Explorer does not require the use of any labels since the microscope directly
measures the refractive index of the different substructures of the cell. However, in the context of
molecular or cellular biology investigations, it can be useful to follow fluorescent markers combined
with the refractive index distribution to validate specific structures or to correlate fluorescent signals
and cellular states.
For this purpose, Nanolive developed the 3D Cell Explorer-fluo (https://nanolive.ch/fluo/) – a
complete solution that combines high precision tomographic data with high quality tri- or fourchannel
epifluorescence provided by a CoolLed module (https://www.coolled.com/).
In this application note we will present the time lapse imaging of mouse embryonic stem cells
(mESCs) that have been genetically modified to express the fluorescence ubiquitination cell cycle
indicator (FUCCI), a two-color (red and green) indicator that allows to monitor the cell cycle phases.
We will explain how to use the 3D Cell Explorer-fluo to record movies that require both fluorescence
and 3D refractive index imaging and will propose a solution to analyze the resulting time lapse
experiment.
Epi-Fluorescence Microscopy: Explore Its Amazing Powers and Uses | The Lifesc...The Lifesciences Magazine
Epi-fluorescence microscopy, also known as epifluorescence microscopy, is a specialized imaging technique that utilizes fluorescence to illuminate specimens of interest.
Lightoptical nanoscopy for the use in biomedical applications and material sciences, detection in attomolar concentrations
* Use of standard fluorophores like GFP, RFP, YFP, Alexa, Fluorescein (no photoswitch necessary)
2CLM Two Color Localisation microscopy in the nanoscale
* Optical resolution 10 nm in 2D, 40 nm in 3D
* Very fast in processing, complete picture (2000 images) with processing in 3 minutes
The basics for symbiosis of Optics and Genetics have been explained in this presentation. " How light can change the very way of life?" .This question has been addressed using relevant web content, consultations from book and through nature videos. This presentation was awarded the highest score in PHM805 at Dayalbagh Educational Institute, Agra.
Shining a light on virus infection with high-resolution microscopyRoland Remenyi, PhD
Each year the Astbury Centre holds a research retreat to promote interdisciplinary activity, to discuss and present latest findings and to promote and encourage interdisciplinary research at the highest level. I communicated my results from a Chikungunya virus project in a 15-min presentation. Slides from this presentation are shown here.
Microdialysis is an integral part of preclinical research to determine extracellular fluid and blood concentrations of metabolites, hormones, drugs, etc, and is often used in quantifying the biochemistry of brain and peripheral tissues. However, it is a molecular-only technique and other imaging modalities are needed to provide the researcher with functional and anatomical information of the animal in vivo.
Optical propagation of blue LED light in brain tissue and Parylene-C used in ...Manjunath Pujar
As a revolutionary neuromodulation technology, optogenetics offers remote manipulation on neural activities of genetically-targeted neural cells with millisecond temporal precision through light illumination. Compared to electrical stimulation, optogenetics has unique benefits including specificity control of neural cell types as well as minimal artifacts and instrumental interferences with electrophysiological recording. Application of optogenetics in neuroscience studies has created an increasing need for the development of light sources and the instruments for light delivery. Among various light sources, micro-light-emitting diodes (μ-LEDs) are favored for its high power efficiency, low cost, and capability of complex system integration. Successful in-vivo optogenetic stimulation on neural cells with the employment of μ-LEDs has been widely reported.
Presentation summarising the 2013 ICSB conference in Copenhagen, a requirement of James Hutton Institute Visits Abroad funding. Presented at the Cellular and Molecular Sciences seminar series.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
The French Revolution, which began in 1789, was a period of radical social and political upheaval in France. It marked the decline of absolute monarchies, the rise of secular and democratic republics, and the eventual rise of Napoleon Bonaparte. This revolutionary period is crucial in understanding the transition from feudalism to modernity in Europe.
For more information, visit-www.vavaclasses.com
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
Ethnobotany and Ethnopharmacology:
Ethnobotany in herbal drug evaluation,
Impact of Ethnobotany in traditional medicine,
New development in herbals,
Bio-prospecting tools for drug discovery,
Role of Ethnopharmacology in drug evaluation,
Reverse Pharmacology.
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
The Indian economy is classified into different sectors to simplify the analysis and understanding of economic activities. For Class 10, it's essential to grasp the sectors of the Indian economy, understand their characteristics, and recognize their importance. This guide will provide detailed notes on the Sectors of the Indian Economy Class 10, using specific long-tail keywords to enhance comprehension.
For more information, visit-www.vavaclasses.com
How to Make a Field invisible in Odoo 17Celine George
It is possible to hide or invisible some fields in odoo. Commonly using “invisible” attribute in the field definition to invisible the fields. This slide will show how to make a field invisible in odoo 17.
19. "Bioluminescent dinoflagellates (Lingulodinium polyedrum) lighting a breaking wave at midnight. The blue light is a result of a luciferase enzyme (like firefly luciferase, but the enzyme in L. polyedrum shares no similarity with that of the firefly enzyme). Under the right conditions, the dinoflagellates become so numerous that the water takes on a muddy reddish color (hence the name "Red Tide"). Image of bioluminescent tide event at a beach in Carlsbad California http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
21. Studies of protein expression using GFP как главный репортерный белок GFP It is possible to report gene expression in single living cells
22. First study describing Green Fluorescent Protein (GFP) as a protein expression marker Science 1994:Vol. 263. no. 5148, pp. 802 - 805 DOI: 10.1126/science.8303295
27. J Biol Chem. 2011 Mar 30. [Epub ahead of print] Mitochondrial dynamics in axons of hippocampal pyramidal neurons
28. llustrated is the photoconversion of a PS-CFP2 fluorescent protein fusion product with human beta -actin using a 405-nanometer diode laser for imaging and conversion, as well as the argon-ion 488-nanometer spectral line for imaging and tracking of the photoconverted protein. P hotoconversion of a PS-CFP2 fluorescent protein as a way to study protein polymers
30. How can we introduce plasmids that are large circular DNA molecules into the cells ? Transfection and infection as methods to introduce gene constructs
39. Rat neocortical neurons infected with recombinant Sindbis virus-enhanced green fluorescent protein in vivo A ) Image of the injection site (arrow) and surrounding area (postnatal day [P] 11). ( B ) Higher magnification images (P 14). Left , layer 2/3 pyramidal neurons. Middle , layer 5 interneuron. Right , layer 5 pyramidal neurons. ( C ) 2-photon laser scanning microscopy image of a layer 2 pyramidal neuron in a section from a mouse (P 36). Lern. Mem. 7, pp. 433-441, 2000 .
40. (a1) Overlay of fluorescent and phase-contrast images illustrating a pair of transfected (green) and nontransfected neurons. (a2) Double whole-cell patch-clamp recording of the pair of neurons shown in (a1). Upper trace shows presynaptic current and lower trace the corresponding postsynaptic current. (b) A pair of neurons both transfected with EGFP. Transfection of hippocampal neurons in low-density microisland cultures
44. Can we study inter- and intramolecular interection in living cells using GFPs? Do we have enough spatial resolution with our conventional microscope systems ?
50. The efficiency of FRET, E FRET , which is defined as probability of the occurrence of energy transfer per donor excitation event, is a steep function of the distance between the fluorophores, r, as given by the following equation: E FRET where Ro is the Forster distance http://www.olympusfluoview.com/applications/fretintro.html
55. The MAG Biosystems™ Dual-View utilizes a single beamsplitter to split the incident beam from the microscope into two independent beams. One beam contains all the emission reflected off of the beamsplitter; the other contains all the emission transmitted through the beamsplitter. Each of these emission channels is projected onto half of the CCD array at exactly the same point in time. Simultaneous multichannel imaging is essential to achieve quantitative emission ratiometric analysis.
57. Cameleon – FRET based Ca 2+ probe Cameleons have been devised by Roger Tsien and others. They are based on fluorescence resonance energy transfer (FRET) between two fluorescent molecules that are linked by a short stretch of calmodulin, a protein that changes its shape in the presence of calcium. In the absence of calcium the two fluorescent proteins are well separated. These calcium sensors are called Cameleons because they change color and have a long tongue (calmodulin) that retracts and extends in and out of its mouth when it binds and releases calcium.
Replica of microscope by Van Leeuwenhoek ~1660 Van Leeuwenhoek's interest in microscopes and a familiarity with glass processing led to one of the most significant, and simultaneously well-hidden, technical insights in the history of science. By placing the middle of a small rod of soda lime glass in a hot flame, Van Leeuwenhoek could pull the hot section apart like taffy to create two long whiskers of glass. By then reinserting the end of one whisker into the flame, he could create a very small, high-quality glass sphere. These spheres became the lenses of his microscopes, with the smallest spheres providing the highest magnifications. An experienced businessman, Leeuwenhoek realized that if his simple method for creating the critically important lens was revealed, the scientific community of his time would likely disregard or even forget his role in microscopy. He therefore allowed others to believe that he was laboriously spending most of his nights and free time grinding increasingly tiny lenses to use in microscopes, even though this belief conflicted both with his construction of hundreds of microscopes and his habit of building a new microscope whenever he chanced upon an interesting specimen that he wanted to preserve. Van Leeuwenhoek used samples and measurements to estimate numbers of microorganisms in units of water. [6] [7] Van Leeuwenhoek made good use of the huge lead provided by his method. He studied a broad range of microscopic phenomena, and shared the resulting observations freely with groups such as the English Royal Society . [8] Such work firmly established his place in history as one of the first and most important explorers of the microscopic world.
Microscopic Section through one year old ash tree ( Fraxinus ) wood, drawing made by Van Leeuwenhoek.
Fluorescence micrographs (low and high magnification) of a neuron co-transfected with mitochondrially-targeted yellow fluorescent protein and cytoplasmic cyan fluorescent protein.
Image of bioluminescent red tide event of 2005 at a beach in Carlsbad California
Progress in transfection technology was relatively slow until the advent of molecular biology techniques for cloning plasmid DNA. These techniques provided the means to prepare and manipulate DNA sequences and the ability to prepare virtually unlimited amounts of relatively pure DNA for transfection experiments. Cloned sequences could also be used to generate RNA in vitro with phage RNA polymerase using DNA templates with the corresponding polymerase promoter (3). As the ability to prepare DNA and RNA for transfection became easier, additional methods, such as electroporation and liposome-mediated transfer, were developed to enable more efficient transfer of the nucleic acids to a broad range of cultured mammalian cells (4,5). The development of reporter gene systems and selection methods for stable gene expression of transferred DNA greatly expanded the applications for gene transfer technology (Figure 1.1). In 1982, Gorman et al. initiated the reporter gene concept with the bacterial chloramphenicol acetyltransferase (CAT) gene and associated CAT assay system (6). Using a reporter gene that is not endogenous to the cell, coupled with a sensitive assay system for that gene product, allows investigators to clone regulatory sequences of interest upstream of the reporter gene to study expression of the reporter gene under various conditions. This technology, together with the availability of transfection reagents, provides the foundation for studying promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein/ protein interactions, translation, and recombination events (7). Since the introduction of the CAT gene and assay system several other reporter systems have been developed for various in vitro and in vivo applications including luciferase, b-galactosidase, alkaline phosphatase and green fluorescent protein (7). See Chapter 6 for detailed descriptions of Promega’s luciferase, CAT and b-galactosidase reporter vectors and assay systems. Integration of DNA into the chromosome, or stable episomal maintenance, of reporter genes and other genes occurs with a relatively low frequency. The ability to select for these cells is made possible using genes that encode resistance to a lethal drug. An example of such a combination is the marker gene for neomycin phosphotransferase with the drug Geneticin® (8). Individual cells that survive the drug treatment expand into clonal groups that can be individually selected, propagated and analyzed. Today the study of gene regulation, the analysis of the expression and function of proteins within mammalian cells, the generation of transgenic organisms and in vivo/ex vivo gene therapy strategies are all made possible by the availability of of gene transfer technologies, nucleic acid molecular biology and reporter gene systems.
The diversity of genetic mutations is illustrated by this San Diego beach scene drawn with living bacteria expressing 8 different colors of fluorescent proteins.
Video 2 (2.9 MB) Mitochondrial dynamics in axons of hippocampal pyramidal neurons: co-transfection with the control, pIRES-GFP Hippocampal neurons were transfected with pDsRed1Mito + pIRES-GFP at 3 DIV. Time lapsed images were obtained of living neurons at 6 DIV capturing an image every 6 seconds for a total of 5 min (total images = 50). The video is shown at 30 x real time.
P hotoconversion of a PS-CFP2 fluorescent protein
Direct microinjection into cultured cells or nuclei is an effective, although laborious technique to deliver nucleic acids into cells. This method has been used to transfer DNA into embryonic stem cells that are used to produce transgenic organisms. However, this technique is not appropriate for studies that require a large number of transfected cells. Electroporation was first reported for gene transfer studies in 1982. This technique is often used for cell types such as plant protoplasts that are particularly recalcitrant to milder methods of gene transfer. The mechanism for entry into the cell is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of nucleic acids into the cell. The technique requires fine-tuning and optimization for duration and strength of the pulse for each type of cell used. A critical balance must be achieved between conditions that allow efficient delivery and conditions that kill cells. Another physical method of gene delivery is biolistic particle delivery . This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells. This method has been successfully employed to deliver nucleic acid to cultured cells, as well as to cells in vivo.
Electroporation was first reported for gene transfer studies in 1982. This technique is often used for cell types such as plant protoplasts that are particularly recalcitrant to milder methods of gene transfer. The mechanism for entry into the cell is based upon perturbation of the cell membrane by an electrical pulse, which forms pores that allow the passage of nucleic acids into the cell. The technique requires fine-tuning and optimization for duration and strength of the pulse for each type of cell used. A critical balance must be achieved between conditions that allow efficient delivery and conditions that kill cells. Another physical method of gene delivery is biolistic particle delivery . This method relies upon high velocity delivery of nucleic acids on microprojectiles to recipient cells. This method has been successfully employed to deliver nucleic acid to cultured cells, as well as to cells in vivo.
Real-time visualization of SCE of TOTO-1 labeled pDsRed2-C1 and subsequent expression of DsRed (A) Overlays of the IR and fluorescence image are shown at the indicated time points (in s) after starting the pulse. The pipette tip was pulled back shortly after pulse application. (B) 24 h after SCE, weak green fluorescence of the TOTO-1 labeled plasmid was visible in the soma. (B ’ ) The same soma in B, visualized by the red fluorescence of DsRed expression. (B ” ) Overlay of B and B ’ . (C) Unlabeled pDsRed2-C1 electroporation leads to strong expression of DsRed. Scalebar in A, 10 mm, in Bƒ, 20 mm, in C, 100 mm.
Transfection Technologies Many transfection techniques have been developed. Desirable features include high efficiency transfer of nucleic acid to the appropriate cellular organelle (for example, DNA into the nucleus), minimal intrusion or interference with normal cell physiology, low toxicity, ease of use, reproducibility, successful generation of stable transfectants, and in vivo efficacy. The techniques developed for gene transfer can be broadly classified as either chemical reagents or physical methods. Chemical Reagents DEAE-dextran was one of the first chemical reagents used for transfer of nucleic acids into cultured mammalian cells (1,9). The ProFection® Mammalian Transfection System-DEAE-Dextran provides reagents for this transfection technique (see Chapter 4 for further information). DEAE-dextran is a cationic polymer that associates with negatively charged nucleic acids. An excess of positive charge, contributed by the polymer in the DNA/polymer complex allows the complex to come into closer association with the negatively charged cell membrane. Uptake of the complex is presumably by of nucleic acids into cells for transient expression; that is, for short-term expression studies of a few days in duration. However, this technique is not generally useful for stable transfection studies that rely upon integration of the transferred DNA into the chromosome (10). Other synthetic cationic polymers have been used for the transfer of DNA into cells, including polybrene (11), polyethyleneimine (12) and dendrimers (13,14). Calcium phosphate co-precipitation became a popular transfection technique following the systematic examination of this method by Graham and van der Eb in the now-classic paper published in 1972 (2). Their study examined the effect of different cations, cationic and phosphate concentrations, and pH on the parameters of transfection. Calcium phosphate co-precipitation is widely used because the components are easily available and reasonable in price, the protocol is easy to use and many different types of cultured cells can be transfected. This method is routinely used for both transient and stable transfection of a variety of cell types. The protocol involves mixing DNA with calcium chloride, adding this in a controlled manner to a buffered saline/phosphate solution and allowing the mixture to incubate at room temperature. This step generates a precipitate that is dispersed onto the cultured cells. The precipitate is taken-up by the cells via endocytosis or phagocytosis. The calcium phosphate also appears to provide protection against intracellular and serum nucleases (15). Promega’s ProFection® Mammalian Transfection System-Calcium Phosphate provides reagents for this transfection technique (see Chapter 4 for further information). By 1980, artificial liposomes were being used to deliver DNA into cells (5). The next advancement in liposomal vehicles was the development of synthetic cationic lipids by Felgner and colleagues (16). Liposome-mediated delivery offers advantages such as relatively high efficiency of gene transfer, ability to transfect certain cell types that are intransigent to calcium phosphate or DEAE-dextran, successful delivery of DNA of all sizes from oligonucleotides to yeast artificial chromosomes (16-20), delivery of RNA (21), and delivery of protein (22). Cells transfected by liposome techniques can be used for transient and for longer term experiments that rely upon integration of the DNA into the chromosome or episomal maintenance. Unlike the DEAE-dextran or calcium phosphate chemical methods, liposome-mediated nucleic acid delivery can be used for in vivo transfer of DNA and RNA to animals and humans (23). A lipid with overall net positive charge at physiological pH is the most common synthetic lipid component of liposomes developed for gene delivery (Figure 1.3). Often the cationic lipid is mixed with a neutral lipid such as L-dioleoyl phosphatidylethanolamine (DOPE) (Figure 1.4). The cationic portion of the lipid molecule associates with the negatively charged nucleic acids, resulting in compaction of the nucleic acid in a liposome/nucleic acid complex. For cultured cells, an overall net positive charge of the liposome/nucleic acid complex generally results in higher transfer efficiencies, presumably because this allows closer association of the complex with the negatively charged cell membrane. Following endocytosis, the complexes appear in the endosomes, and later in the nucleus. It is unclear how the nucleic acids are released from the endosomes and traverse the nuclear membrane. DOPE is considered a “fusogenic” lipid (24) and it is thought that its role may be to release these complexes from the endosomes, as well as to facilitate fusion of the outer cell membrane with the liposome/nucleic acid complexes. Promega provides a variety of transfection reagents that use cationic lipids for the delivery of nucleic acids to eukaryotic cells. These include TransFast™ Transfection Reagent, the Tfx™ Reagents and Transfectam® Reagent. See Chapter 3 for more information on the use of these reagents. endocytosis. This method is successful for delivery
Flow chart of the main procedures of Ca2+ phosphate transfection protocol
Formation of optimal DNA/Ca2+-phosphate precipitate and subsequent dissolution to stop transfection (a) Continuous vortexing when mixing DNA with Ca2+ and phosphate buffer results in large clusters of precipitate. The precipitate was examined after 1 h incubation. (b, c) Formation of optimal DNA/Ca2+-phosphate precipitate achieved by gently vortexing the DNA/Ca2+ solution with phosphate buffer. Image taken after 1 h incubation. (d) Dissolving precipitate with slightly acidic transfection medium preequilibrated in a 10% CO2 incubator in order to reduce its toxicity to neurons. Scale bar, 50 mm.
Transmission electron micrograph of multiple bacteriophages attached to a bacterial cell wall Virus entry requires sequential interaction between specific viral membrane glycoproteins and cellular receptors. Much of the recent work elucidating these receptors and the viral glycoproteins interacting with them has been carried out in the laboratories of P. Spear (Northwestern University), and G. Cohen and R. Eisenberg of the University of Pennsylvania. Upon entry the nucleocapsid is transported to the nuclear pores, where viral DNA is released into the nucleus. The viral genome is accompanied by the a-TIF protein which functions in enhancing immediate early viral transcription via cellular transcription factors. The virion-associated host shutoff protein ( vhs --UL41) appears to remain in the cytoplasm where it causes the disaggregation of polyribosomes and degradation of cellular and viral RNA.
Rat neocortical neurons infected with recombinant Sindbis virus-enhanced green fluorescent protein in vivo imaged in fixed tissue sections
Transfection of hippocampal neurons in low-density microisland cultures facilitates electrophysiological analysis
Hippocalcin translocation in dendritic shaft was assessed by FRET between HPCA1-14-CFP and HPCA-YFP. A demonstrates a part of dendritic tree where hippocalcin translocation was observed as a result of network bursting. Hippocalcin fluorescence changes and FRET efficiency in ROIs shown in red and green in A are demonstrated in B and C. E, F and G. Translocation changes follows changes in FRET indicating that local plasma membrane hippocalcin association is a reason for the translocation. H. An electrical activity simultaneously recorded in the neuron in a cell-attached configuration. D. Averaged changes (over 16 pairs of red and neighboring green ROIs) in hippocalcin fluorescence and FRET efficiency. I. The same set of sites engaged in hippocalcin signaling possibly indicating distribution of hippocalcin membranous targets.
A real-time movie of confocal real color images and corresponding pseudocolored ratio images showing Ca2+ waves inside cells, which were evoked by histamine, in HeLa cells expressing YC3.60(Yellow Cameleon). The images were taken at video rate (30 Hz) using a color 3CCD camera for simultaneous acquisition of CFP (cyan-emitting mutant of GFP) and YFP (yellow-emitting mutant of GFP) images. To improve z axis resolution, a spinning disk confocal unit was placed in front of the camera. Ten micromolar histamine was added to the recording medium to activate receptor-evoked Ca2+ release from endoplasmic reticulum.
Design and plasma membrane expression of VSFP2s A: A pair of CFP (donor) and YFP (acceptor) is attached to the 4-transmembrane-voltage-sensing domain (VSD) of Ci-VSP. B, C: Confocal fluorescence (B) and transmission images (C) of PC12 cells transfected with VSFP2D. Note the targeting of the fluorescent protein to the plasma membrane. Scale bar is 30 μm.