방법의 원리 : 이 사이트는 전체 plasmids의 돌연변이 유발이 동영상에 직접 플라스미드로 몇 기지의 대체, 삭제 또는 삽입하여 복제된 대상 유전자를 변경할 수있는 돌연변이 유발 방법이 설명 감독. 그것은 비 PCR 기반 thermocycling 반응에서 전체 플라스미드를 확장하여 사용할 수 있습니다. 반응하는 동안 원래의 플라스미드에 불일치의 형태로 원하는 돌연변이를 운반 mutagenic primers는, 새로 합성 플라스미드에 통합되어 있습니다. 반응에서 원래 플라스미드의 제거 후 변이 플라스미드가 E.으로 변화 도착 이 방법의 돌연변이 효율이 100 % 없기 때문에 대장균. 다음 단계만을 심사 목적으로하고 있습니다. 전체 절차는 사흘 걸리지만 주요 부분은 하루 이내에 할 수 있습니다. 1.0 하루에 한 : 1.1 Thermocycling 반응 : 원본 플라스미드 : 돌연변이 유발 프로토콜을 감독이 사이트 10킬로바이트까지 plasmids와 함께 잘 작동됩니다. 큰 Plasmids이 방법으로 공격하는 조금 어려운 일부 인내와 thermocycling 조건 및 / 또는 관할 세포의 조절이 걸릴 수 있습니다. 또한 당신이 작업하는 플라스미드는 댐 + 박테리아 스트레인으로부터 격리해야합니다. thermocycling 반응을 위해 당신이가 자신을 공격하려는 플라스미드의 10-60 ngs 필요합니다. Primers : 당신 thermocycling 반응을 설정할 수 전에 손에에서 primers을 가지고있다. 당신은 모든 mutagenic 입문서 약 150 NG가 필요합니다. 당신이 1시 10분 희석 100 PM 주식의 1.5 μl을한다면 그것은 괜찮습니다. 귀하의 mutagenic primers를 디자인할 때 고려해야 할 몇 가지 간단한 지침이있다 : <ul><li> primers 서로 보완되어야 </li><li> primers 길이 25 45 세포핵 사이 여야합니다 </li><li> 원래 플라스미드에 불일치 형태의 변이는 두 primers에 포함되어 있어야합니다 </li><li> 불일치는 뇌관 중심으로 각 면에 최소한 8 세포핵 둘러싸인해야 </li><li> primers 적어도 40 % GC - 내용이 있어야합니다 </li><li> primers는 하나 이상의 거시기 뭐냐 또는 고사 5 총리 3 프라임을 종료해야합니다 </li><li> primers는 phosphorylated 필요가 없습니다 없으며 그들은 단지 그들이해야 desalted FPLC 또는 페이지 정화,해야 할. </li><li> TM의 계산에 대해 어떤 수식하지만 배송 인증서 TM이 60 이상되어야 ° C. 필요하지 않습니다 </li><li> 심사 목적을 위해 그것은 당신의 돌연변이와 제한 사이트를 삽입하거나 삭제하는 실용적입니다. 때문에 유전자의 변성의 원하는 돌연변이와 제한 사이트를 삽입할 수있는 많은 가능성이 있습니다. 뉴잉글랜드 Biolabs의 웹사이트는 적절한 제한 사이트를 찾을 수있는 도구를 제공합니다. 간단히 DNA에 대한 모호함 코드를 사용하여, 당신이 원하는 아미노산 서열에 대한 코딩하여 프라이머 시퀀스를 입력합니다. 효소 찾기 도구는 제한 사이트가 원하는 돌연변이에 평행 도입 수있는 당신에게 말할 것이다. 이 방법으로 모든 가능한 한 실제적인 제한 사이트를 찾을 수 없다면, 당신은 돌연변이의 사이트에있는 유전자에 관한 새로운 제한없이 사이트를 삽입할 수 있습니다. 이후이 제한 사이트가 새로운 mutagenic primers 삽입에 의해 삭제됩니다되는 돌연변이 시리즈를위한 템플릿으로이 플라스미드 사용할 수 있습니다. 당신이 플라스미드 같은 사이트에서 돌연변이의 시리즈를 할 계획이라면이 방법은 매우 편리합니다. </li></ul> DNA - 효소 :이 방법 여러분은 3&#39; - 5 &#39;Exonuclease 활동을 전시하고 뭉툭한 끝을 만들어 내열성 DNA 중합 효소 - 필요합니다. 우리는 항상 Fermentas에서 재조합 Pfu - 중합 효소를 사용합니다. 당신이 증폭 단계에 문제가있다면 당신은 높은 품질의 효소를 시도 수도 있지만 대부분의 목적에 표준 Pfu 충분합니다. dNTPs, 효소 버퍼와 물로 반응을 완료합니다. 1.2 Thermocycling 조건 thermocycler은 다음과 같은 방법으로 설정되어야합니다. 초기 및 반복 변성 단계는 95 ° C.에 30sec로 설정됩니다 primers의 어닐링 온도는 복잡한 수식 계산해서는 안됩니다. 우리는 일반적으로 55 ° C 및 1 분 어닐링 시간 어닐링 온도를 설정합니다. 당신이 대부분 사용합니다 25-30 세포핵의 primers 경우, 이것은 우리의 시간의 95 %를 작동합니다. 60 ° C. -이 일을하지 말아야하는 경우 여하튼, 간단하게 50 사이의 어닐링 온도를 변화 시도 신장 온도는 사용하는 효소에 따라 달라집니다. 이 예제에서 우리는 72 신장 온도 요구 Fermentas에서 Pfu - 효소 °​​ C.를 사용하여 연신율 시간은 플라스미드의 크기에 따라 다릅니다. 우리는 항상 킬로바이트 당 1 분을 계산하고, 그 한 분 추가 추가시간. 예를 들어, 9킬로바이트 플라스미드에 대해 우리는 연신율 시간 10 분 선택한 것입니다. 18 사이클 정도 추가로 사용하기 위해 플라스미드 변이 만드는 데 충분합니다. 낮은 사이클의 수를 유지, 또한 시간을 절약할 수 있습니다. <img src="/files/ftp_upload/1135/figure1.png" alt="그림 1" width="600" height="287" /> Thermocycling 반응 후 1.3 Gelcheck 성공적인 확대는 전기 영동을 수행하여 확인한다. 자전거가 완료 직접 후, 1 %의 태 아가로 오스 겔에 반응 5 μl를로드합니다. 증폭이 성공했다면, 당신은 별개의 밴드를 확인할 수 있습니다. 새로 합성된 DNA가 젤에 명확하게 표시되지 않으면 그러나 전체 반응을 촉진하려고하고 다음 단계에서 변환을 위해 사용할 수 있습니다. 우리를 위해 이것은 거의 효과가 없어 가지고 있지만 사용해 가치가있다. 반응이 작동하지 않을 경우에 가장 좋은 것은 어닐링 온도를 조절하는 것입니다. 1.4 DpnI의 소화 : 변환하기 전에 템플릿으로 제공하는 원래의 플라스미드가 강한 배경을 방지하기 위해 반응에서 제거되어야합니다. 이 DpnI로 제한 소화에 의해 이루어집니다. 이 제한 endonuclease는 플라스미드 methylated 상처. A는 methylated되어야 반면, 그 인식 및 제한 사이트는 시퀀스 GATC입니다. DpnI와 소화 경우에만 댐에서 고립되었던 원래 unmutated 플라스미드가 + 변형이 탈락, methylated되지 않은 새로 합성 변이 플라스미드는 DpnI에 의해 영향을받지 않습니다. 단순히 반응 DpnI 1-2 μl를 추가하고 37에서 적어도 한 시간을 품어 °. 빠른 다이제스트 DpnI을 사용할 때 배양 시간은 15 분 감소 수 있습니다. 귀하의 DpnI이 제한 소화의 배양 시간의 품질이 크게 unmutated 플라스미드와 배경이되는 방법을 강한 결정합니다. 1.5 변환 : DpnI는 소화 후 플라스미드는 유능한 E.으로 변환을위한 준비 대장균 세포. 간단히 관할 세포에 DpnI의 소화 반응에서 5 μl을 추가하고 실험실에서 권장하는대로 변환을 수행합니다. 우리 사건에서 우리는 30 분 얼음 박테리아 - 플라스미드 혼합 잠복기 후 42 ° C 90 초에서 열 충격 그걸로 열 충격 절차를 사용합니다. 200 μl SOC - 솔루션을 추가하면, 박테리아는 적극적으로 한 H. 위해 37, ° C를 흔들어, incubated 아르 일시간 후 세균은 한천 미디어를 선택을 도금하고 있습니다. 2.0 하루에 두세 클론 1 부 2.1 심사 : 클론의 선택 돌연변이 효율이 100 %가 아니므로 귀하의 돌연변이에 대한 화면으로 필요합니다. 우리는 우리가 추가하거나 뇌관 통합을 통해 삭제된 제한 사이트의 존재 또는 부재를 확인하는 제한 소화하여이 작업을 수행할. 이러한 목적을 위해 우리는 보통 여덟 식민지를 선택하고 다음날 플라스미드 준비 밤새도록 그들을 성장. 3.0 지나고 3 클론 2 부 3.1 상영 : 마커 효소로 제한 소화 셋째 날에는 당신의 마커 효소와 미니 준비와 후속 제한 소화를 수행합니다. 겔에서는 다음 원하는 돌연변이를 운반하고있는 사람 아닌 클론 어느 확인할 수 있습니다. 제한 소화를 사용하는 심사 방법은 잘 작동합니다. 그럼에도 불구하고, 당신은 시퀀싱하여 성공적인 돌연변을 확인합니다.

<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.

사이트 감독 돌연변이 유발이 플라스미드에 의해 운반 유전자를 공격하는 빠른 방법을 제공하는 돌연변이 유발 - 방법입니다. 전체 반응은 하루에 할 수 있습니다. 이 방법을 사용하면, 그것은 단지 원하는 변이를 가지고 무료 primers 한 쌍의 등 Pfu - 효소로 교정 효소가 필요합니다. 새로 합성된 플라스미드는 제한 효소 DpnI와 반응을 소화하여 부모의 플라스미드 분리 수 있습니다. 이 효소는 methyla...

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

전체 plasmids의 사이트 이동 돌연변이 유발은 원래 플라스미드의 약간 다른 유사 콘텐츠를 작성하는 간단한 방법입니다. 이 방법으로 복제된 대상 유전자 직접 플라스미드로 몇 기지의 대체, 삭제 또는 삽입하여 변경할 수 있습니다. 그것은 단순히이 아닌 PCR 기반 thermocycling 반응에서 전체 플라스미드를 확장하여 사용할 수 있습니다. 반응 동안 원하는 돌연변이를 운반 mutagenic primers는, 새로 합성 플라스미드에 통합되어 있습니다. 이 비디오 튜토리얼에서 우리는 플라스미드에 기본 재료를 소개하는 쉽고 비용 효과적인 방법을 보여줍니다. 프로토콜은 표준 시약 작동 종종 매우 비싼 상용 키트에서 독립적입니다. 이 프로토콜을 적용하는 것은 그것이 상업 키트의 일부를 사용하는 비용은 무엇 여덟째에 대한 반응의 총 비용을 줄일 수 있습니다. 이 비디오에서 우리는 또한 과정에서 중요한 단계에 대해 언급하고 mutagenic primers를 디자인하는 방법에 대한 자세한 지침을 제공합니다.

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

Homemade Site Directed Mutagenesis of Whole Plasmids

전체 plasmids의 사이트 이동 돌연변이 유발은 원래 플라스미드의 약간 다른 유사 콘텐츠를 작성하는 간단한 방법입니다. 여기서 우리는 플라스미드를 사용 표준 시약에 기본 재료를 소개하는 쉽고 비용 효과적인 방법을 보여줍니다.

전체 Plasmids의 수제 사이트 감독 돌연변이 유발

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 조회수.  Johannes Gutenberg-University Mainz, Germany. 전체 plasmids의 사이트 이동 돌연변이 유발은 원래 플라스미드의 약간 다른 유사 콘텐츠를 작성하는 간단한 방법입니다. 여기서 우리는 플라스미드를 사용 표준 시약에 기본 재료를 소개하는 쉽고 비용 효과적인 방법을 보여줍니다.

비디오: 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.

특정 사이트 Recombinase (SSR) 기술의 원리

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

연구

생물학

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.

이온 채널의 돌연변이 유발과 기능 분석 Heterologously 포유 동물 세포에 표현

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.

웜 실험실 소개 : Culturing 웜에서 돌연변이 유발하는

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.

에서 단백질의 감독 진화에 대한 돌연변이 유발 및 기능 선택 프로토콜 E. 대장균</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.

막 단백질의 Conformational 역학 검사 현장에서 사이트 이동 형광 라벨링과

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.

편광 상피 세포에 Plasmids의 Microinjection에 의해 작은 GTPases의 기능을 분석

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.

단일 Oocyte의 Bisulfite의 돌연변이 유발

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.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

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.

생성<em&gt; 엔테로 박터</em&gt; SP. YSU 영양 요구는 트랜스포존 돌연변이 유발을 사용하여

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.

높은 처리량 시퀀싱을 활용하여 전체 - 단백질 포화 돌연변이 유발 도서관의 기능 평가를위한 프로토콜

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