Das Prinzip der Methode: Die Mutagenese des gesamten Plasmide erklärt in diesem Video ist eine Mutagenese-Methode, die Sie zu einem geklonten Zielgen durch Substitution, Deletion oder Insertion von wenigen Basen direkt in ein Plasmid ändern können. Es funktioniert durch die Verstärkung der gesamten Plasmid, in einem nicht PCR-basierte Thermocycling Reaktion. Während der Reaktion mutagene Primer, die die gewünschten Mutation in Form von Fehlanpassungen der ursprünglichen Plasmid, werden in die neu synthetisierte Plasmid integriert. Nach dem Entfernen des ursprünglichen Plasmid aus der Reaktion des mutierten Plasmid wird in E. verwandelt coli. Die folgenden Schritte sind für Screening-Zwecke nur, weil die Mutation Effizienz dieser Methode ist nicht 100%. Die ganze Prozedur dauert drei Tage, aber der größte Teil innerhalb eines Tages durchgeführt werden. 1,0 Tag eins: 1,1 Thermocycling Reaktion: Original-Plasmid: Diese Mutagenese-Protokoll funktioniert am besten mit Plasmiden bis zu 10kb. Größere Plasmide sind ein bisschen schwierig, mit dieser Methode mutieren und etwas Geduld und Anpassung der Thermocycling Bedingungen und / oder der zuständigen Zellen. Außerdem das Plasmid, das Sie mit muss von einem Damm + Bakterienstamm isoliert werden. Für die Thermocycling Reaktion, die Sie benötigen 10 bis 60 ngs des Plasmids Sie mutieren. Grundierungen: Bevor Sie einrichten können Ihre Thermocycling Reaktion müssen Sie Ihre Primer zur Hand haben. Sie benötigen etwa 150 ng eines jeden mutagene Primer. Es ist ok, wenn man 1,5 ul einer 1:10 verdünnten 100 pM Bilanz zu ziehen. Es gibt ein paar einfache Richtlinien, die Sie bei der Gestaltung Ihrer mutagene Primer muss: <ul><li> Die Primer sollten komplementär zueinander </li><li> Die Primer sollten zwischen 25 und 45 Nukleotide lang sein </li><li> Die Mutationen in Form von Fehlanpassungen der ursprünglichen Plasmid muss in beide Primer enthalten sein </li><li> Das Missverhältnis sollte in der Grundierung zentriert und flankiert von mindestens 8 Nukleotiden auf jeder Seite </li><li> Die Primer sollten eine GC-Gehalt von mindestens 40% </li><li> Die Primer sollten 5 prime und 3 prime mit einem oder mehreren Gs oder Cs Ende </li><li> Die Primer müssen nicht phosphoryliert werden, noch müssen sie FPLC oder PAGE gereinigt, lediglich entsalzt sie sein sollten. </li><li> Für die Berechnung der Tm Sie brauchen keine Formel, sondern Tm auf den Versand-Zertifikat sollte höher sein als 60 ° C. </li><li> Für Screening-Zwecke ist es praktisch, einfügen oder löschen Restriktionsstelle mit Ihrem Mutation. Aufgrund der Degeneration des genetischen Codes gibt es viele Möglichkeiten, um eine Restriktionsschnittstelle mit Ihrem gewünschten Mutation einfügen. Die Website des New England Biolabs bietet ein Werkzeug, um eine solche geeignete Restriktionsschnittstelle zu finden. Geben Sie einfach Ihre Primer-Sequenz kodierend für die Aminosäuresequenz Sie es wünschen, mit der Mehrdeutigkeit Code für DNA. Das Enzym-Finder-Tool wird Ihnen sagen, welche Restriktionsstellen parallel zu Ihrem gewünschten Mutation eingeführt werden. Wenn Sie nicht finden können, eine mögliche oder praktische Restriktionsstelle durch diese Weise können Sie neue Restriktionsschnittstelle ohne Bezug auf den genetischen Code an der Stelle der Mutation einfügen. Danach können Sie mit diesem Plasmid als Template für eine Reihe von Mutationen in dem dieser Restriktionsstelle durch Einfügung des neuen mutagene Primer gelöscht werden. Dieser Ansatz kann sehr praktisch, wenn Sie planen, eine Reihe von Mutationen an der gleichen Stelle des Plasmids zu tun haben. </li></ul> DNA-Polymerase: Für dieses Verfahren benötigen Sie eine thermostabile DNA-Polymerase, die 3&#39;-5 &#39;Exonuklease-Aktivität aufweist und schafft stumpfen Enden. Wir verwenden immer rekombinante Pfu-Polymerase von Fermentas. Wenn Sie Probleme mit der Verstärkung Schritte haben Sie vielleicht versuchen, eine höhere Qualität Polymerase, aber für die meisten Zwecke der Standard Pfu genug sein. Vervollständigen Sie die Reaktion mit dNTPs, Polymerase-Puffer und Wasser. 1,2 Thermocycling Bedingungen Die Thermocycler sollte in folgender Weise eingestellt werden. Die erste und wiederkehrende Denaturierung Phase ist es, 30sec bei 95 ° C eingestellt Die Annealingtemperatur der Primer darf nicht mit komplizierten Formeln berechnet werden. Wir in der Regel legen Sie die Annealingtemperatur auf 55 ° C und die Glühzeit zu einer Minute. Für Primer 25 bis 30 Nukleotiden, die Sie verwenden werden meistens funktioniert dies für uns 95% der Zeit. Jedenfalls, wenn dies nicht funktionieren sollte, versuchen Sie einfach die Variation der Annealing-Temperatur zwischen 50 - und 60 ° C. Die Dehnung ist abhängig von der Polymerase die Sie benutzen. In dieser Demonstration verwenden wir die Pfu-Polymerase von Fermentas, die für eine Dehnung Temperatur von 72 ° C nennt Die Dehnung der Zeit variiert je nach Größe des Plasmids. Wir sind immer zu berechnen 1 min pro kb, und fügen Sie eine zusätzliche Minute an, dassZeit. Zum Beispiel für eine 9kb Plasmid wir wählen würden 10min als Dehnung der Zeit. 18 Zyklen ausreichend sind, um zu schaffen genug mutierte Plasmid für die weitere Verwendung. Keeping die Anzahl der Zyklen gering ist, spart auch Zeit. <img src="/files/ftp_upload/1135/figure1.png" alt="Abbildung 1" width="600" height="287" /> 1,3 Gelcheck nach thermischer Reaktion Die erfolgreiche Amplifikation sollte mittels einer Elektrophorese überprüft werden. Direkt nach dem Radfahren beendet ist, laden 5 ul der Reaktion auf ein 1% TAE-Agarosegel. Wenn die Verstärkung erfolgreich war, sollten Sie eine deutliche Bande. Allerdings, wenn das neu synthetisierte DNA ist nicht deutlich sichtbar auf dem Gel, können Sie versuchen, Niederschlag der ganzen Reaktion und verwenden es für die Transformation in den nächsten Schritt. Für uns hat nur selten gearbeitet, aber es lohnt sich zu versuchen. Das Beste, was zu tun ist, wenn die Reaktion nicht funktioniert ist die Glühtemperatur anzupassen. 1,4 DpnI Verdauung: Vor Transformation der ursprünglichen Plasmid, das als Vorlage diente müssen aus der Reaktion auf starken Hintergrund zu verhindern entfernt werden. Dies wird durch Restriktionsverdau mit DpnI getan. Diese Restriktionsendonuklease schneidet nur methylierte Plasmid. Seine Anerkennung und Restriktionsstelle wird die Sequenz GATC, während A hat methyliert werden. Wenn Verdau mit DpnI nur das Original nicht mutierten Plasmid, das von einem Damm isoliert + Stamm geschnitten wird, ist das neu synthetisierte mutierte Plasmid, das nicht methylierte nicht durch DpnI betroffen. Einfach geben Sie 1-2 pl DpnI, um die Reaktion und inkubieren sie mindestens eine Stunde bei 37 °. Bei Verwendung des Schnell verdauen DpnI die Inkubationszeit kann bis etwa 15 Minuten verringert werden. Die Qualität Ihrer DpnI und die Inkubationszeit dieser Restriktionsverdau stark bestimmt, wie stark Ihr Hintergrund mit mutierten Plasmid wird. 1,5 Transformation: Nach DpnI Verdauung des Plasmids ist bereit für die Umwandlung in die zuständige E. coli-Zellen. Fügen Sie einfach 5 pl vom DpnI Abbaureaktion in die kompetenten Zellen und die Transformation wie in Ihrem Labor empfohlen. In unserem Fall verwenden wir das Hitzeschock-Verfahren durch Inkubation der Bakterien-Plasmid-Mischung auf Eis für 30 min und dann Hitzeschock es bei 42 ° C für 90 sek. Nach Zugabe von 200 ul SOC-Lösung werden die Bakterien inkubiert, kräftiges Schütteln bei 37 ° C für 1 h. Nach 1 Stunde werden die Bakterien auf die Auswahl-Agar ausplattiert. 2,0 Tag zwei 2.1 Abschirmung der Klone Teil 1: Auswahl der Klone Da die Mutation Effizienz ist nicht 100% Sie brauchen, um für Ihre Mutanten Bildschirm. Wir tun dies durch Restriktionsverdau, in denen wir prüfen für An-oder Abwesenheit der Restriktionsschnittstelle, die wir hinzugefügt oder gelöscht durch Primer-Integration. Zu diesem Zweck haben wir in der Regel abholen acht Kolonien und wachsen sie über Nacht für Plasmid-Präparation auf den nächsten Tag. 3,0 Tag drei 3,1 Screening der Klone Teil 2: Restriktionsverdau mit Markerenzym Am dritten Tag Sie eine Mini Prep und anschließende Restriktionsverdau mit Ihrem Markerenzym. Auf dem Gel können Sie dann sehen, welche Ihrer Klone tragen die gewünschte Mutation und welche nicht. Das Screening-Verfahren mit Restriktionsverdau funktioniert sehr gut. Dennoch sollten Sie Ihre erfolgreiche Mutation durch Sequenzierung bestätigt.

<ol>
	<li>Papworth, C., Bauer, J. C., Braman, J., Wright, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=QuikChange+site-directed+mutagenesis.">QuikChange site-directed mutagenesis.</a> Strategies. 9, 3-4 (1996).</li></ol>

The authors have nothing to disclose.

Seite Mutagenese ist eine Mutagenese-Verfahren, das eine schnelle Möglichkeit, ein Gen von einem Plasmid durchgeführt mutieren zur Verfügung stellt. Die gesamte Reaktion kann in nur einem Tag durchgeführt werden. Mit dieser Methode wird es brauchen nur ein Paar kostenlose Primer, die die gewünschten Mutationen und das Korrekturlesen Polymerase wie Pfu-Polymerase. Die neu synthetisierte Plasmid kann von den Eltern Plasmid durch Verdauen der Reaktion mit dem Restriktionsenzym DpnI getrennt werden. Dieses Enzym verdau...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Pfu polymerase (recombinant or native)</td>
<td>&nbsp;</td>
<td>fermentas</td>
<td>EP0571 or EP0501</td>
<td>&nbsp;</td>
<tr>
<td>dNTP mix 10 mM </td>
<td>&nbsp;</td>
<td>fermentas</td>
<td>R0191</td>
<td>&nbsp;</td>
<tr>
<td>DpnI or DpnI Fast digest</td>
<td>&nbsp;</td>
<td>fermentas</td>
<td>ER1701</td>
<td>&nbsp;</td>
<tr>
<td>primers</td>
<td>&nbsp;</td>
<td>invitrogen</td>
<td>&nbsp;</td>
<td>desalted</td>
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homemade site directed mutagenesis of whole plasmids

Seite Mutagenese ganzer Plasmide ist ein einfacher Weg, um etwas anders Variationen eines Original-Plasmid zu erzeugen. Mit dieser Methode wird die geklonte Zielgen kann durch Substitution, Deletion oder Insertion von wenigen Basen direkt in ein Plasmid verändert werden. Es funktioniert einfach durch die Verstärkung der gesamten Plasmid, in einem nicht PCR-basierte Thermocycling Reaktion. Während der Reaktion mutagene Primer, die das gewünschte Mutation in die neu synthetisierte Plasmid integriert. In diesem Video-Tutorial zeigen wir eine einfache und kostengünstige Möglichkeit, Basensubstitutionen in ein Plasmid einzuführen. Das Protokoll arbeitet mit Standard-Reagenzien und ist unabhängig von kommerziellen Kits, die oft sehr teuer. Anwendung dieses Protokolls können die Gesamtkosten einer Reaktion auf ein Achtel von dem, was es kostet mit einigen der kommerziellen Kits reduzieren. In diesem Video, das wir auch auf kritischen Schritte Kommentar während des Prozesses und geben detaillierte Anweisungen, wie die mutagene Primer-Design.

Watch this Scientific Journal Video about Homemade Site Directed Mutagenesis of Whole Plasmids at JoVE.com

Homemade Site Directed Mutagenesis of Whole Plasmids

Seite Mutagenese ganzer Plasmide ist ein einfacher Weg, um etwas anders Variationen eines Original-Plasmid zu erzeugen. Hier zeigen wir eine einfache und kostengünstige Möglichkeit, Basensubstitutionen in ein Plasmid mit Standard-Reagenzien einzuführen.

Hausgemachte Mutagenese von Whole Plasmide

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

homemade-site-directed-mutagenesis-of-whole-plasmids

Research

JoVE Journal

Biology

33.0K Aufrufe.  Johannes Gutenberg-University Mainz, Germany. Seite Mutagenese ganzer Plasmide ist ein einfacher Weg, um etwas anders Variationen eines Original-Plasmid zu erzeugen. Hier zeigen wir eine einfache und kostengünstige Möglichkeit, Basensubstitutionen in ein Plasmid mit Standard-Reagenzien einzuführen.

Serie: Homemade Site Directed Mutagenesis of Whole Plasmids

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Principles of ortsspezifische Rekombinase (SSR)-Technologie

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Forschung

Biologie

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenese und funktionale Analyse von Ionenkanälen heterolog in Säugerzellen exprimierten

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

An Introduction to Worm Lab: from Culturing Worms to Mutagenesis

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane sulfonate (EMS) and 4, 5', 8-trimethylpsoralen combined with ultraviolet light (TMP/UV). Nematodes are powerful biological systems for genetics studies because of their simple body plan and mating system, which is composed of self-fertilizing hermaphrodites and males that can generate hundreds of progeny per animal. Nematodes are maintained in agar plates containing a lawn of bacteria and can be easily transferred from one plate to another using a pick. EMS is an alkylating agent commonly used to induce point mutations and small deletions, while TMP/UV mainly induces deletions. Depending on the species of nematode being used, concentrations of EMS and TMP will have to be optimized. To isolate recessive mutations of the nematode Pristionchus pacificus, animals of the F2 generation were visually screened for phenotypes. To illustrate these methods, we mutagenized worms and looked for Uncoordinated (Unc), Dumpy (Dpy) and Transformer (Tra) mutants.

Screening for mutants with phenotypic defects is a straightforward method for identifying genes that function in a given biological process. In this article we describe how to culture free living worms (e.g., Pristionchus pacificus) in the laboratory and show two different mutagenesis methods, EMS and TMP/UV.

An Introduction to Worm Lab: von Kultivierung Worms zu Mutagenese

This protocol describes procedures to maintain nematodes in the laboratory and how to mutagenize them using two alternative methods: ethyl methane ...

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

Mutagenese und Functional Selection Protocols for Directed Evolution von Proteinen in E. coli</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Die Untersuchung der Konformationsänderungen Dynamics von Membranproteinen In situ Mit Site-directed Fluoreszenzmarkierung

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Analyzing the Function of Small GTPases by Microinjection of Plasmids into Polarized Epithelial Cells

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces the 'free' surfaces and the basolateral membrane is in contact with the substrate and neighboring cells. Both membrane domains are separated by tight junctions, which form a diffusion barrier. Apical-basolateral polarization can be recapitulated successfully in culture when epithelial cells such as Madin-Darby Canine Kidney (MDCK) cells are seeded at high density on polycarbonate filters and cultured for several days 1 2. Establishment and maintenance of cell polarity is regulated by an array of small GTPases of the Ras superfamily such as RalA, Cdc42, Rab8, Rab10 and Rab13 3 4 5 6 7. Like all GTPases these proteins cycle between an inactive GDP-bound state and an active GTP-bound state. Specific mutations in the nucleotide binding regions interfere with this cycling 8. For example, Rab13T22N is permanently locked in the GDP-form and thus dubbed 'dominant negative', whereas Rab13Q67L can no longer hydrolyze GTP and is thus locked in a 'dominant active' state 7. To analyze their function in cells both dominant negative and dominant active alleles of GTPases are typically expressed at high levels to interfere with the function of the endogenous proteins 9. An elegant way to achieve high levels of overexpression in a short amount of time is to introduce the plasmids encoding the relevant proteins directly into the nuclei of polarized cells grown on filter supports using microinjection technique. This is often combined with the co-injection of reporter plasmids that encode plasma membrane receptors that are specifically sorted to the apical or basolateral domain. A cargo frequently used to analyze cargo sorting to the basolateral domain is a temperature sensitive allele of the vesicular stomatitis virus glycoprotein (VSVGts045) 10. This protein cannot fold properly at 39&deg;C and will thus be retained in the endoplasmic reticulum (ER) while the regulatory protein of interest is assembled in the cytosol. A shift to 31&deg;C will then allow VSVGts045 to fold properly, leave the ER and travel to the plasma membrane 11. This chase is typically performed in the presence of cycloheximide to prevent further protein synthesis leading to cleaner results. Here we describe in detail the procedure of microinjecting plasmids into polarized cells and subsequent incubations including temperature shifts that allow a comprehensive analysis of regulatory proteins involved in basolateral sorting.

This article details the procedures involved in overexpression and analysis of small GTPases in polarized epithelial cells using microinjection technique.

Die Analyse der Funktion von kleinen GTPasen durch Mikroinjektion von Plasmiden in polarisierten Epithelzellen

Epithelial cells polarize their plasma membrane into biochemically and functionally distinct apical and basolateral domains where the apical domain faces ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Einzel Oocyte Bisulfit-Mutagenese

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can be generated using a transposome. The commercially available, Tn5-derived transposome used in this protocol consists of a linear segment of DNA containing an R6K&#947; replication origin, a gene for kanamycin resistance and two mosaic sequence ends, which serve as transposase binding sites. The transposome, provided as a DNA/transposase protein complex, is introduced by electroporation into the prototrophic strain, Enterobacter sp. YSU, and randomly incorporates itself into this host&#8217;s genome. Transformants are replica plated onto Luria-Bertani agar plates containing kanamycin, (LB-kan) and onto M-9 medium agar plates containing kanamycin (M-9-kan). The transformants that grow on LB-kan plates but not on M-9-kan plates are considered to be auxotrophs. Purified genomic DNA from an auxotroph is partially digested, ligated and transformed into a pir+ Escherichia coli (E. coli) strain. The R6K&#947; replication origin allows the plasmid to replicate in pir+ E. coli strains, and the kanamycin resistance marker allows for plasmid selection. Each transformant possesses a new plasmid containing the transposon flanked by the interrupted chromosomal region. Sanger sequencing and the Basic Local Alignment Search Tool (BLAST) suggest a putative identity of the interrupted gene. There are three advantages to using this transposome mutagenesis strategy. First, it does not rely on the expression of a transposase gene by the host. Second, the transposome is introduced into the target host by electroporation, rather than by conjugation or by transduction and therefore is more efficient. Third, the R6K&#947; replication origin makes it easy to identify the mutated gene which is partially recovered in a recombinant plasmid. This technique can be used to investigate the genes involved in other characteristics of Enterobacter sp. YSU or of a wider variety of bacterial strains.

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Enterobacter sp. YSU grows in glucose minimal salts medium. Auxotrophs are generated by transforming it with a transposome which randomly inserts itself into the host genome. Mutants are found by replica plating from complex medium to minimal medium. Interrupted genes are identified by gene rescue and sequencing.

Erzeugung<em&gt; Enterobacter</em&gt; Sp. Ysu Auxotrophe Mit Transposon-Mutagenese

Prototrophic bacteria grow on M-9 minimal salts medium supplemented with glucose (M-9 medium), which is used as a carbon and energy source. Auxotrophs can ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

Ein Protokoll für Functional Assessment of Whole-Protein Sättigungsmutagenese-Bibliotheken mit Hilfe von High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...