Name: preparation of raav9 to overexpress or knockdown genes in mouse hearts
Uploaded: 2016-12-17T13:00:00
Description: Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

We thank Dr. Zaffar Haque for careful reading of the manuscript. We thank Drs. Masaharu Kataoka and Gengze Wu for discussions and help. Work in the Wang lab is supported by the American Heart Association, Muscular Dystrophy Association, and NIH (HL085635, HL116919, HL125925).

Alle beschreven stappen werden uitgevoerd in het kader van protocollen door de bioveiligheid Comite en de Institutional Animal Care en gebruik Comite van Boston Children&#39;s Hospital goedgekeurd. Boston Children&#39;s Hospital heeft pathogenen vrije muis faciliteiten met gereguleerde licht / donker cycli en klimaatregeling. Veterinaire en dierenverzorgers verandering kooien en zorgen voor de gezondheid van de muizen. De faciliteiten zijn AAALAC gecertificeerd en hebben actief Dierenwelzijn Assurance certificering (AAA...

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Het is belangrijk om ongewenste ITR recombinatie te minimaliseren tijdens plasmideconstructie. Vóór het genereren van het virus, moet men altijd controleren de ITR integriteit van de AAV plasmiden met restrictie digestie en agarosegelelektroforese. Het is onmogelijk om 100% intacte plasmiden te verkrijgen, maar de verhouding recombinatie moet zoveel mogelijk worden beperkt. Minder dan 20% aanvaardbaar is voor een succesvolle rAAV9 verpakking. Opmerkelijk, kweken van de bacteriën bij lagere temperatuur (30 ° C) met e...

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.
One of the authors names and affiliation was corrected from:
Jian-Ming Jiang3,4 
3 Department of Genetics, Harvard Medical School 
4 Howard Hughes Medical Institute
to:
Jianming Jiang5 
5 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore

A correction was made to the Authors section in: <a href="http://www.jove.com/video/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts</a>.

Erratum: Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Regelen expressie of activiteit van specifieke genen in verschillende biologische systemen is een waardevol strategie het onderzoek van genfunctie 1. Een directe manier van de verwezenlijking van dit doel is om nucleotidesequenties te manipuleren en het genereren van mutante allelen. Hoewel het maken van nauwkeurige, doelgerichte wijzigingen aan het genoom van levende cellen nog een tijdrovend en arbeidsintensief praktijk, de ontwikkeling van de machtige TALEN en CRISPR / Cas9 gereedschap heeft een nieuw tijdperk van het genoom bewerken 2-5 geopend. Een routine laboratorium methode voor genetische manipulatie heeft zich gericht op de introductie van genetisch materiaal (DNA en RNA&#39;s die coderende sequenties of siRNA / shRNAs) in de cellen uit te drukken of knockdown het gen (s) van belang 1,6. In veel gevallen, de belangrijke hinderpaal voor genmanipulatie is de afgifte van DNA, RNA of eiwit in de cellen. Wat in vitro studies, efficiënt transfectiop systemen zijn vastgesteld in veel gekweekte cellijnen. In het muismodel met name in vivo genafgifte is moeilijker. Er zijn een aantal extra- en intracellulaire barrières die moeten worden gepasseerd om efficiënte cellulaire opname van exogeen reagens bereiken. Extra obstakels onder meer de snelle klaring en de korte duur van de geleverde materialen 7,8. Een strategie om deze problemen te omzeilen is om virale vectoren als &quot;dragers&quot; of &quot;auto&quot; in vivo genafgifte. De natuur geëvolueerd transductie eigenschappen van virussen mogelijk maken de efficiënte levering van een gen van belang in cellen 7,9,10. Talrijke types van virale vectoren zijn ontwikkeld en maken flexibele in vivo genmanipulatie in verschillende organen en cellen in muizen. De meest gebruikte virale systemen omvatten Retrovirus, Lentivirus, adenovirus en adeno-geassocieerde virus (AAV) <sup&gt; 11. Retrovirussen zijn enkelstrengs RNA-virussen en hun genetisch materiaal naar het gastheergenoom op stabiele wijze gedurende mitose voeren, waardoor nog levenslange expressie van het getransduceerde genen in de doelwitcellen en organen 12-14. Veel soorten retrovirussen alleen delende cellen infecteren en hun doeltreffendheid in niet-delende cellen zeer laag 15. Dit beperkt hun nut voor gen-levering. Lentivirus is een geslacht van de familie Retroviridae. Anders dan andere retrovirussen, Lentivirus kan zowel delende als niet-delende cellen infecteren en is op grote schaal gebruikt voor genoverdracht in post-mitotisch en zeer gedifferentieerde cellen 16. De levenscyclus van Lentivirus omvat ook de integratie van vector DNA in het gastheergenoom. Aldus Lentivirus-gemedieerde genafgifte worden stabiele en langdurige expressie van het getransduceerde genetische elementen 16-18. Dit kan echter voorzien van een double-e vertegenwoordigendged zwaard in het gebruik van deze virussen om genexpressie te manipuleren, de integratie van vector DNA kan leiden tot mutagenese in de gastheercellen en kunnen artefact veroorzaken. Adenovirus andere veelgebruikte genafgiftesysteem. Anders dan retrovirussen en lentivirussen, adenovirussen zijn niet geïntegreerd en niet interfereren met de genomische integriteit van gastheercellen 8,10,11,19. Bovendien kunnen adenovirussen transfecteren DNA in vele celtypen, en infectie niet afhankelijk actieve celdeling 19. Een ander belangrijk kenmerk van adenovirussen is het gemak van vector zuivering als virale vectoren de mogelijkheid te repliceren 19,20. De belangrijkste nadeel van dit systeem is dat adenovirus infectie kan leiden tot sterke immuunresponsen in doelwitcellen en organen 19, beperkt het gebruik ervan in verschillende onderzoeken, in het bijzonder bij gentherapie studies. Vergeleken met deze verschillende typens van virale vectoren, wordt recombinant adeno-geassocieerd virus (rAAV) naar het ideale genafgiftesysteem 21,22 zijn. Het vertoont een minimale immunogeniciteit en pathogeniciteit 23,24. Bovendien rAAV infecteert een breed scala aan celtypen, waaronder zowel verdelen en niet-delende cellen. In de meeste gevallen is rAAV niet te integreren in ontvangst genomen; waardoor het risico van ongewenste genetische of genomische veranderingen in de doelcellen laag 22. Recentelijk rAAV systemen met succes gebruikt voor de in vivo afgifte van DNA coderend voor eiwitten, miRNAs, shRNAs en CRISPR-gRNAs in muis hartspier 23,25-29. Deze methode heeft fundamenteel onderzoek en gentherapie studies vergemakkelijkt het gebied van cardiovasculair onderzoek. Hier, de gedetailleerde procedure om rAAV9 vectoren die efficiënt overexpressie of knockdown de genen van belang in muisharten werd beschreven te genereren. Het protocol voorziet in een eenvoudige en effectieve methodemanipuleren van cardiale genexpressie in muizen experimentele modellen.

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			<td height="21" style="height:21px;width:575px;">Polyethylenimine, Linear (MW 25,000)</td>
			<td style="width:241px;">Polysciences, Inc.&#160;</td>
			<td style="width:192px;">#23966-2</td>
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			<td height="21" style="height:21px;width:575px;">Tube, Polypropylene, 36.2 mL, 25 x 87 mm, (qty. 56)</td>
			<td style="width:241px;">Beckman Coulter, Inc</td>
			<td style="width:192px;"># 362183</td>
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			<td height="21" style="height:21px;width:575px;">Nuclease, ultrapure</td>
			<td style="width:241px;">SIGMA</td>
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			<td height="21" style="height:21px;width:575px;">Density Gradient Medium(Iodixanol)</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#D1556-250ML</td>
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			<td height="21" style="height:21px;width:575px;">Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td style="width:241px;">EMD Millipore Corporation</td>
			<td style="width:192px;">#UFC910008</td>
			<td></td>
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			<td height="21" style="height:21px;width:575px;">Laboratory pipetting needle with 90&#176; blunt ends,gauge 14, L 6 in., nickel plated hub</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">#CAD7942-12EA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Poloxamer 188 solution (Pluronic&#174; F-68 solution)</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">P5556-100ML</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Proteinase K</td>
			<td style="width:241px;">SIGMA</td>
			<td style="width:192px;">3115828001</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:575px;">DNase I</td>
			<td style="width:241px;">Roche</td>
			<td style="width:192px;">10104159001</td>
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			<td height="21" style="height:21px;width:575px;">Centrifuge machine</td>
			<td style="width:241px;">Thermo Scientific</td>
			<td style="width:192px;">75004260</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Centrifuge System</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:192px;">363118</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Ultracentrifuge</td>
			<td style="width:241px;">Beckman Coulter</td>
			<td style="width:192px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">DMEM medium</td>
			<td style="width:241px;">Fisher Scientific</td>
			<td style="width:192px;">SH30243FS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">Fetal Bovine Serum&#160;</td>
			<td style="width:241px;">Atlanta Biologicals&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:192px;">S11150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:575px;">rAAV9 vector</td>
			<td style="width:241px;">Penn Vector Core</td>
			<td style="width:192px;">P1967</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of raav9 to overexpress or knockdown genes in mouse hearts

Die de expressie of activiteit van specifieke genen door myocardiale afgifte van genetisch materiaal in muriene modellen maakt het onderzoek van genfuncties. Hun therapeutisch potentieel in het hart kan worden bepaald. Er zijn beperkte benadering voor in vivo moleculaire interventie in het muizenhart. Recombinant adeno-geassocieerd virus (rAAV) gebaseerde genoom techniek is gebruikt als een essentieel instrument voor in vivo cardiale genmanipulatie. De specifieke voordelen van deze technologie zijn onder andere een hoge efficiëntie, hoge specificiteit, lage genomische integratie rate, minimale immunogeniciteit en weinig pathogeniciteit. Hier wordt een gedetailleerde procedure voor de bouw, verpakking, en zuiveren het rAAV9 vectoren beschreven. Subcutane injectie van rAAV9 in neonatale pups resulteert in robuuste expressie of efficiënte knockdown van het gen (en) van belang in het muizenhart, maar niet in de lever en andere weefsels. Met de cardiale specific TNNT2 promotor, hoge expressie van GFP-gen in het hart werd verkregen. Daarnaast richten mRNA werd geremd in het hart wanneer een rAAV9-U6-shRNA werd gebruikt. Praktische kennis van rAAV9 technologie kunnen nuttig zijn voor hart- en onderzoeken zijn.

De strategieën voor rAAV9 bouw van rAAV9.cTNT :: GFP of rAAV9.U6 :: shRNA plasmiden worden getoond in figuren 1 en 2, respectievelijk. Zoals de voorbeelden werd de rAAV9 vector gegenereerd om het GFP gen tot overexpressie in muisharten. Het resulterende plasmide bevat de cTnT :: GFP geflankeerd door twee ITR plaatsen (Figuur 1). De rAAV9.U6 :: shRNA vector werd geconstrueerd voor knockdown Trbp mRNA (figuur 2) <p cl...

Watch this Scientific Journal Video about Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts at JoVE.com

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

Voorbereiding van de rAAV9 tot overexpressie of Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

The overall goal of this procedure is to prepare recombinant adeno-associated virus nine, or rAAV9, to manipulate gene expression in mouse hearts. The method can answer key question in the field of cardiology and molecular genetics, such as how to study gene function in mouse heart in vivo. The main advantage of this technique is that it will allow us to easily manipulate the gene expression and function in vivo in mouse heart, allowing the functional access of gene function without having to rely on the genetically modified animal models.After generating rAAV9 constructs and culturing HEK293 cells according to the text protocol, to transfect the cells on day one, combine 70 micrograms of AAV Rep-Cap plasmid, 70 micrograms of rAAV9 plasmid, and 200 micrograms of Ad helper plasmid in a 50-milliliter centrifuge tube. Add 49 milliliters of room temperature DMEM without FBS to the tube and mix well. Then, to make a four-to-one PEI to DNA solution, add 1, 360 microliters of PEI to the tube and mix well.Incubate the solution at room temperature for 15 to 30 minutes. After the incubation, add five milliliters of the solution to each 150-milliliter dish of cells. Culture the cells at 37 degrees Celsius and 5%CO2 for 50 to 72 hours.To harvest the transfected cells, dislodge and suspend the cells by using the culture medium to pipette up and down. Then transfer the cell suspensions to sterile 50-milliliter tubes. Centrifuge the cells at 500 times G for five minutes.Then add five milliliters of PBS to each tube and re-suspend the cell pellets before combining all the suspensions in one 50-milliliter tube. After spinning the cells at 500 times G for five minutes, discard the supernatant. To purify the AAV, re-suspend the pellet in 10 milliliter of lysis buffer.Freeze the lysate at minus 80 degrees Celsius or in a dry ice ethanol bath. Then thaw it at 37 degrees Celsius. Vortex the lysate for one minute before repeating the freeze and thaw two more times.Next, add one millimolar magnesium chloride to the thawed lysate. Then add nuclease to a final concentration of 250 units per milliliter. Incubate the solution at 37 degrees Celsius for 15 minutes to dissolve the DNA protein aggregation.Centrifuge the sample at 4, 800 times G and four degrees Celsius for 20 minutes. Then collect the supernatant. Meanwhile, to prepare a 17%iodixanol gradient solution, mix five milliliters of 10X PBS with 0.05 milliliters of one molar magnesium chloride, 0.125 milliliters of one molar potassium chloride, 10 milliliters of five molar sodium chloride, and 12.5 milliliters of density gradient medium.Use water to adjust the total volume to 50 milliliters. After preparing 25, 40, and 60%gradient solutions according to the text protocol, use a needle and syringe to load five milliliters of 17%gradient solution into a polypropylene tube. Follow this with five milliliters each of 25, 40, and 60%gradient solutions.Layer the lysate on top of the gradient. Then fill the tube with lysis buffer and cover it with a cork. Centrifuge the gradient at 185, 000 times G and 16%Celsius for 90 minutes.Following the spin, use a syringe to harvest the 40%viral fraction by inserting a 21 gauge needle into the intersection between the 40 and 60%fractions, completely avoiding the 25%layer. Mix the viral fraction with sterilized polyoxyethylene polyoxypropylene, or PEG-PPG block copolymer PBS solution, up to a total volume of 15 milliliters. Load the mixture into a filter tube with a molecular weight cutoff of 100 kilodaltons.Then centrifuge the sample at 2, 000 times G and four degrees Celsius for 30 minutes. Decant the solution from the bottom of the tube. Then use PEG-PPG to refill the filter tube to 15 milliliters.Centrifuge the tube at 2, 000 times G and four degrees Celsius for 20 minutes. After repeating the refilling and spin two more times, collect the purified rAAV9 virus fraction still above the filter. Aliquot 100 to 400 microliters of the purified rAAV9 into 1.7-milliliter tubes, depending on the viral titer, and store the virus at minus 80 degrees Celsius.After determining the viral titer according to the text protocol, prepare rAAV9 working solutions with virus titers of one to seven times 10 to the 12th particles per milliliter. To treat neonatal mice, use the rAAV9 solution to pre-fill a 0.33 by 12.7-milliliter insulin syringe with a 29 gauge one-half inch needle, avoiding air bubbles. After cryo-anesthetizing a pup, hold the pup in one hand with thumb and forefinger, then swipe the back skin of the pup with a swab stick saturated with 70%isopropyl alcohol.Insert the syringe into the anterior dorsal subcutis of the animal at an angle of five to 10 degrees before injecting 50 to 70 microliters of the rAAV9 solution. The standard curve for rAAV9 titration was generated with the qPCR data by linear regression. The manipulated variable y represents the log10 value of the DNA molecular concentration of each standard sample, and the corresponding variable x represents the Ct value.The log10 concentration values and Ct numbers exhibit a nice linear correlation and fit with the equation shown here. Titers of rAAV9 samples were calculated based on the linear equation. Using the method demonstrated in this video, a high titer of rAAV9 vectors was obtained in this representative study.To monitor the efficiency and tissue specificity of rAAV9 cTNT vectors, P0.5 pups were treated with the same amount of rAAV9 cTNT luciferase or rAAV9 cTNT GFP by subcutaneous injection. Two weeks after injection, robust expression of GFP was detected in the heart but not in other organs. To monitor the knockdown efficiency and tissue specificity of rAAV9 U6 vectors, P0.5 mice were treated with the same amount of rAAV9 U6 scramble or rAAV9 U6 Trbp shRNA by subcutaneous injection.Two weeks after injection, the mRNA level of Trbp in the heart was substantially reduced by rAAV9 U6 shTrbp. Down-regulation of Trbp was also detected to a lesser extent in the liver. Once mastered, rAAV preparation can be done in five days, if it is formulated properly.While attempting this procedure, it is important to remember to use healthy 293 cells for rAAV packaging. After its development, this technique paved the way for researchers in the field of cardiology and genetics to explore gene functions and gene therapeutic potentials in mouse hearts. After watching this video, you should have a good understanding of how to prepare recombinant AAV9 to manipulate the gene expressing any function in mouse hearts.

preparation-of-raav9-to-overexpress-or-knockdown-genes-in-mouse-hearts

Research

JoVE Journal

Genetics

The Use of Induced Somatic Sector Analysis (ISSA) for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree&#39;s lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant&#39;s secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

The Use of Induced Somatic Sector Analysis ISSA for Studying Genes and Promoters Involved in Wood Formation and Secondary Stem Development

Here we present a protocol that facilitates the medium to high throughput functional characterization of gene and promoter constructs in tree secondary stem tissue within comparatively short time frames. It is efficient, easy to use and widely applicable to a range of tree species.

Het gebruik van Induced Somatic Sector Analysis (ISSA) voor het bestuderen van genen en Promoters Betrokken bij Wood Vorming en secundaire Stem Development

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little ...

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

-Peptide afgeleide methode om genen en eiwitten Across Cellulaire en organellen Barriers in Planten Transport

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Grootbrengen en Double-stranded RNA-gemedieerde gen knock-down in het verbergen Kever,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting mutant alleles reside at their endogenous genomic sites and no exogenous DNA sequences are left in the genome following the procedure. Since in haploid yeast cells each of the four core histone proteins is encoded by two non-allelic genes with highly homologous open reading frames (ORFs), targeting mutagenesis specifically to one of two genes encoding a particular histone protein can be problematic. The strategy we describe here bypasses this problem by utilizing sequences outside, rather than within, the ORF of the target genes for the homologous recombination step. Another feature of this system is that the regions of DNA driving the homologous recombination steps can be made to be very extensive, thus increasing the likelihood of successful integration events. These features make this strategy particularly well-suited for histone gene mutagenesis, but can also be adapted for mutagenesis of other genes in the yeast genome.

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

A strategy for generating mutations in histone genes at their endogenous location in Saccharomyces cerevisiae is presented.

gerichte<em&gt; In Situ</em&gt; Mutagenese van histon genen in gist

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

Voorbijgaande expressie van buitenlandse genen in Insect cellen (sf9) voor eiwit functionele Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

Gelijktijdige Video-EEG-ECG controle om te identificeren Neurocardiac dysfunctie in muismodellen van epilepsie

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

qKAT: kwantitatieve Semi-automated te typen van Killer-cel immunoglobuline-achtig Receptor genen

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

Een in vitro protocol voor het evalueren van MicroRNA-niveaus, functies en geassocieerde doel genen in tumor cellen

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic screening using chemical mutagens is a useful strategy for mapping mutant phenotypes to genes among Drosophila and other model organisms that share conserved cellular pathways with humans. If the mutated gene of interest is not lethal in early developmental stages of flies, a climbing assay can be conducted to screen for phenotypic indicators of decreased brain functioning, such as low climbing rates. Subsequently, secondary histological analysis of brain tissue can be performed in order to verify the neuroprotective function of the gene by scoring neurodegeneration phenotypes. Gene mapping strategies include meiotic and deficiency mapping that rely on these same assays can be followed by DNA sequencing to identify possible nucleotide changes in the gene of interest.

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

We present a protocol using a forward genetic approach to screen for mutants exhibiting neurodegeneration in Drosophila melanogaster. It incorporates a climbing assay, histology analysis, gene mapping and DNA sequencing to ultimately identify novel genes related to the process of neuroprotection.

In vivo genetisch scherm naar voren om nieuwe neuroprotectieve genen in Drosophila melanogaster te identificeren

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Modulation of Tau Subcellular Localization as a Tool to Investigate the Expression of Disease-related Genes

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, recent evidence indicated that Tau is present also in other subcellular compartments including the nucleus where it is implicated in DNA protection, in rRNA transcription, in the mobility of retrotransposons and in the structural organization of the nucleolus. We have recently demonstrated that nuclear Tau is involved in the expression of the VGluT1 gene, suggesting a molecular mechanism that could explain the pathological increase of glutamate release in the early stages of Alzheimer&#39;s disease. Until recently, the involvement of nuclear Tau in modulating the expression of target genes has been relatively uncertain and ambiguous due to technical limitations that prevented the exclusion of the contribution of cytoplasmic Tau or the effect of other downstream factors not related to nuclear Tau. To overcome this uncertainty, we developed a method to study the expression of target genes specifically modulated by the nuclear Tau protein. We employed a protocol that couples the use of localization signals and the subcellular fractionation, allowing the exclusion of the interference from the cytoplasmic Tau molecules. Most notably, the protocol is easy and is composed of classic and reliable methods that are broadly applicable to study the nuclear function of Tau in other cell types and cellular conditions.

Tau is a neuronal protein present both in the cytoplasm, where it binds microtubules, and in the nucleus, where it exerts unconventional functions including the modulation of Alzheimer&#39;s disease-related genes. Here, we describe a method to investigate the function of nuclear Tau while excluding any interferences coming from cytoplasmic Tau.

Modulatie van Tau Subcellulaire lokalisatie als een instrument om de uitdrukking van ziektegerelateerde genen te onderzoeken

Tau is a microtubule binding protein expressed in neurons and its main known function is related to the maintenance of cytoskeletal stability. However, ...