T.R.R conceived the project, designed and performed experiments and prepared the manuscript. E.J.K and S.E.M performed experiments. All authors contributed intellectually to the final manuscript. This work was funded by a University of Leicester Innovation Fellowship to SEM and by a UK Medical Research Council (MRC) Senior Research Fellowship to S.M.C (MR/J009202/1). DNA sequencing was performed by Protein and Nucleic Acid Laboratories (PNACL), Centre for Core Biotechnology Services, University of Leicester. We wish to thank A. Francis Stewart for providing the Rox and Dre plasmids. pL451 and pL452 plasmids were obtained from National Cancer Institute (NCI), Frederick, USA.

<ol>
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T.R.R is the inventor of a British patent application on multiplex recombineering technology.

بناء خطوط الخلايا ES ونماذج الماوس وشمل تاريخيا استهداف الجينات باستخدام يبني البلازميد التي تحتوي على أليل تعديل 4. ومع ذلك، فقد ثبت أن بناء هذه النواقل التي تستهدف الجينات المعقدة ليكون عنق الزجاجة كبير في إنتاج مثل هذه النماذج في الوقت المناسب. وقد سمح وضع استراتيجيات بناء ناقلات recombineering أساس تصاميم ناقلات تحسين والتجمع ناقلات أكثر كفاءة. ومع ذلك، بروتوكولات recombineering الحالية لا تزال تنطوي على خطوات متعددة، تتطلب تنقية البلازميد المتوسطة واستخدام سلالات بكتيرية مختلفة. استنساخ فرعي الإدراج زائد يقدم نهجا جديدا لبناء ناقلات التي يمكن القيام بها في حالة Electroporation للواحد في سلالة المضيف BAC المقيمين. تم اختبار فائدة SPI في استهداف الجين هنا في مجموعة متنوعة من التطبيقات بناء ناقلات الأمراض. في جميع الحالات درست هنا، وثبت SPI لتكون فعالة وتم إنتاج البلازميد المؤتلف الصحيح.في الغالبية العظمى من الحالات، تم إدراج أشرطة مختلفة متعددة بشكل صحيح في ناقلات الاستهداف. أشرطة DNA الكبيرة وناقلات تم استيعابها بسهولة في بروتوكول SPI وأظهرت مرونة هذا النظام. يعتمد SPI على استخدام تسلسل تناظر طويلة وphosphorothioate (PTO) حماية أشرطة DNA الخطية. تعديل PTO يمنح حماية ضد exonucleases على الحمض النووي الخطي 20،21 وHA طويلة يزيد من كفاءة إعادة التركيب للسماح المتنوعة. ومع ذلك، والتوليف من تسلسل بنسبة ضئيلة أطول يزيد من فرص تراكم الأخطاء خصوصا الحذف. الطفرات في جزئية يمكن أن يكون ضارا ولا سيما إذا كانوا تغطية المناطق البروتين الترميز. ينصح بشدة تسلسل الحمض النووي عبر HA وتغطية طول كامل من الكاسيت إدراجها للقضاء على الحيوانات المستنسخة مع أي تعديلات التسلسل. ويقترح استخدام نظام البلمرة DNA عالية الدقة أيضا لتجنب إدخالأخطاء ذ PCR. طول وتكوين HA من البلازميد استنساخ فرعي هو أكثر أهمية بالنسبة لتلك التي الكاسيت الإدراج (لا تظهر البيانات). وبالنسبة للتطبيقات ذات حساسية خاصة مثل بناء ناقلات knockin، حيث لا يتم التسامح مع أي طفرات في المناطق اكسون، وHA من الكاسيت الإدراج يمكن اختصارها (50-120 بي بي) لتفادي المشاكل المرتبطة oligos طويلة. ويمكن أيضا الكاسيت الإدراج أن تترك phosphorothioated معدلة أو المزدوجة (حيث معرفة اتجاه تكرار غير متوفرة). ولكن مضاعفة في هذه الحالات لا يزال يتطلب محمية لفترة طويلة HA البلازميدات استنساخ فرعي. والتحذير من هذه الاستراتيجية معينة هو خفض كفاءة المتنوعة التي يمكن أن تؤثر SPI الاستنساخ في مواضع مختلفة. طول كاسيت الإدراج والبلازميد هو استنساخ فرعي معلمة هامة أخرى في SPI الاستنساخ. جزيئات DNA أكبر electroporate أقل كفاءة وتأثير تراكمي، نظرا لإعادةquirement لتقديم كافة الأشرطة في نفس الخلية (لا تظهر البيانات). مضاعفة هو الأكثر كفاءة مع أشرطة الإدراج أصغر. DNA شظايا أكبر من 3 كيلوبايت أيضا وضع حد على معالجة ssDNA الأحمر بوساطة، والتي هي الأكثر كفاءة تصل إلى 3 كيلو بايت 30. أشرطة الإدراج تتجاوز 3 كيلو بايت هي مزدوجة مقطوعة وتمتزج أقل بكفاءة عن طريق بيتا مسار مستقل 20. لذلك، وفحص المستعمرات كافية لتحديد استنساخ الصحيح يصبح من المهم في هذه الحالات. مدة أطول من إعادة التركيب آخر Electroporation لليزيد أيضا من فرص الشفاء من استنساخ الصحيح في التدريبات SPI الصعبة. ما يصل إلى أربعة أشرطة صغيرة (&lt;1.5 كيلوبايت) يمكن إدراجها في وقت واحد مع عملية SPI، على الرغم من كفاءة مضاعفة تتناقص مع كل كاسيت إضافية (لا تظهر البيانات). ومع ذلك، وهذا يعكس نتائج تجربة SPI الأمثل ويستحسن النظر في حدود المتنوعة عند استخدام العديد من أشرطة كبيرة. </P&gt; تطوير أدوات التحرير الجينوم مثل تتجمع interspaced بانتظام يكرر المتناوب قصيرة (كريسبر) نظام نوكلياز داخلية -cas9 وقد مكن إنشاء التعديلات الجينوم جديدة وأدى إلى زيادة كفاءة الهندسة الجينوم 36. ومع ذلك، فقد استكملت هذه التكنولوجيات الجديدة الجينات التي تستهدف ناقلات بدلا من حلت محل لهم. ومن المتوقع أن النظام كريسبر-CAS يمكن أن تحل محل اختيار المضادات الحيوية وزيادة صقل بروتوكول recombineering متعدد.

وقد مكن تطوير تقنيات استهداف الجين بناء خطوط الخلايا ونماذج الماوس للتحقيق في الأنظمة البيولوجية المختلفة 1-3. والمرحلة الأولى الأساسية في تعديل تسلسل الجيني هو تصميم وبناء الجينات التي تستهدف ناقلات 4. النواقل التي تستهدف هي يبني البلازميد التي تحمل أليل من الفائدة التي تحتوي على التعديلات المرغوبة (ق) يحيط بها علامة الاختيار (على سبيل المثال، النيومايسين)، والمناطق الجينومية الطويلة اللازمة لكفاءة إعادة التركيب مثلي في خلايا الثدييات 5. ويتحقق الدقيق تعديل الجينات من خلال إدخال متجه إلى استهداف الجذعية الجنينية (ES) خلايا 6 أو 7 الخلايا الجسدية، حيث إعادة التركيب مثلي بين مساحات مماثلة من تسلسل الحمض النووي على ناقلات الاستهداف والنتائج موضع الجينومية في نقل تعديل المقصود إلى الجينوم عن طريق تحويل الجينات 8. هذا modifiخلايا ES إد يمكن حقنها في الكيسات الأريمية الماوس لإنتاج نسل (chimaeras) التي يمكن بعد ذلك نقل هذه الأليلات المعدلة عبر الجرثومية 9. طرق بديلة لإنتاج الفئران المعدلة وراثيا ينطوي على Microinjection من متجه التعبير الجيني في واحدة بيضات ملقحة الماوس الخلية، مما يؤدي إلى التكامل عشوائية من متجه في جينوم الفأر 10. وقد اعتمدت الطرق التقليدية في البناء على ناقلات التقليدية &quot;قص ولصق&quot; الاستنساخ باستخدام أنزيمات التقييد وligases DNA لاستنساخ علامة الاختيار مختلفة وشظايا الجينومية في العمود الفقري النواقل. ومع ذلك، عاملا يحد المتأصل في الاستنساخ التقليدي هو تحديد المواقع واختيار مواقع التقييد، وخاصة مع تسلسل الحمض النووي أطول. وغالبا ما يتطلب خطوات استنساخ فرعي متعددة وأيضا يدخل تسلسل الحمض النووي غريبة في ناقلات، الذي غالبا ما يؤدي إلى انخفاض كفاءة من استهداف الجينات. Recombineering (ENG recombinogenicineering) هو تقنية الهندسة DNA أن يتغلب على هذه القيود باستخدام إعادة التركيب مثلي (HR) بوساطة البروتينات فج إعادة التركيب في E. خلايا القولونية 11،12. منذ أن أي منطقة من تسلسل مثلي بمثابة ركيزة من recombineering، تتم إزالة القيود من توافر مواقع قيود. تسلسل الحمض النووي كبيرة يمكن تعديلها بسهولة مباشرة في الجسم الحي، وبالتالي الحفاظ على السلامة الهيكلية أيضا على 13. Recombineering فعال جدا مع homologies قصيرة (50 نقطة) ويمكن إدراج 14 وبالتالي الأسلحة التماثل (HA) مريح في تسلسل بنسبة ضئيلة الاصطناعية. في تجربة نموذجية recombineering، وبنسبة ضئيلة أو مزدوج DNA الذين تقطعت بهم السبل (dsDNA) جزء يحتوي على HA هو electroporated إلى recombineering المختصة E. خلايا القولونية التي تحتوي على الهدف تقع إما على كروموسوم أو على البلازميد 15. تمنح إمكانيات إعادة التركيب من قبل التعبير محرض للتفصيل الأحمرombination البروتينات من فج 16،17 أو البروتينات RecET من طليعة العاثية RAC 18. ونوكلياز خارجية الأحمر / RECE تحويل dsDNA الخطية إلى DNA واحد الذين تقطعت بهم السبل (ssDNA) وسيطة، والتي يتم بعد ذلك ملزمة شريكتها الأحمر / مصحح، وهو بروتين الصلب واحد الذين تقطعت بهم السبل (SSAP) 19. والصلب من ssDNA طويلة أو قصيرة بنسبة ضئيلة لسلسلتها الهدف مكملة يحدث على حبلا متخلفة من شوكة تكرار ويؤدي إلى إدراج تسلسل في الموقع المستهدف. متخلفة حبلا ssDNA إعادة التركيب هو أساس كفاءة عالية من recombineering ويمكن وصفها من قبل &#39;بيتا&#39; نموذج إعادة التركيب 20،21. وسير العمل recombineering نموذجي لبناء ناقلات استهداف الجين ينطوي على أي من طريقين التالية. واحد الطريق ينطوي على استنساخ فرعي المنطقة الجينومية المرجوة من استنساخ فأر BAC إلى البلازميد يليه الإدراج متتابعة من المواقع إعادة التركيب LoxP، علامة الاختيار <sتصل&gt; 22 الخ، أو الطريق البديل ينطوي على استهداف BAC موضع الجيني مع مختلف عناصر مكافحة ناقلات استهداف من قبل جولات متعددة من recombineering 10،23 ثم استنساخ فرعي موضع المعدلة في البلازميد بواسطة إصلاح الفجوة استنساخ 24،25. وقد استخدمت الاختلافات حول هذا الموضوع في مختلف خطوط أنابيب عالية الإنتاجية recombineering كجزء من برامج إنتاج الماوس كبير 26،27. ومع ذلك، تنطوي هذه الإجراءات مراحل معقدة وطويلة، تتطلب استخدام ناقلات المتخصصة وE. القولونية سلالات (على سبيل المثال، لجنة المساواة العرقية معربا عن الخلايا) والاستفادة من واحد أو أكثر من الخطوات الوسيطة من متجه تنقية الحمض النووي وإعادة التحول (الجدول 1). استنساخ فرعي الإدراج زائد (SPI) هو أسلوب recombineering الرواية التي تجمع بين إعادة التركيب بيتا وإصلاح الفجوة الاستنساخ في عملية واحدة (الشكل 1). SPI التجمع ناقلات بسيط، سريع ومرن ويوفر تحسنا كبيرا على recombi القياسيةنهج neering (الجدول 1). هنا، علينا أن نظهر سهولة وجدوى استخدام SPI لمختلف التطبيقات بناء ناقلات مع التركيز بشكل خاص على بناء تصاميم غير القياسية وتحدي النواقل. وتشمل حالات الاختبار بناء الفلورسنت مراسل knockin ناقلات، مزدوج الموسومة التعبير البروتين ناقلات knockin، وهو BAC الفلورسنت مراسل النواقل وبالضربة القاضية ناقلات مشروط. هذه دراسة بأشكال مختلفة شرط لحصر مستقبلات سطح الخلية، تنقية مجمع البروتين النووي أو يجتذ مشروط التعبير عن الجين.

<table border="0" cellpadding="0" cellspacing="0" width="1597">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Name of Material/ Equipment</td>
			<td style="width:127px;">Company</td>
			<td style="width:216px;">Catalog Number</td>
			<td style="width:941px;">Comments/Description</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:315px;">Antibiotics except Zeocin</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">Ampicillin, A9518-5G; 
			Chloramphenicol , C1919-5G; Blasticidin, 15205-25MG; Gentamicin, G1264-50MG; Hygromycin, H3274-50MG; Kanamycin,: K1876-1G; 
			Tetracycline hydrochloride, T7660-5G; Trimethoprim, 92131-1G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Zeocin</td>
			<td style="width:127px;">Invivogen:&#160;</td>
			<td style="width:216px;">ant-zn-1</td>
			<td>Zeocin is also available from Life technologies/Fisher</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">C57BL/6 BAC clones</td>
			<td style="width:127px;">CHORI: https://bacpac.chori.org/&#160;</td>
			<td style="width:216px;">RPCI-23 or RPCI-24 BAC libraries</td>
			<td>129/Sv BAC clones can be obtained from Invivogen or Life technologies/Fisher</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">KOD hotstart DNA polymerase system</td>
			<td style="width:127px;">Millipore: https://www.merckmillipore.com</td>
			<td style="width:216px;">71086-3</td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:315px;">L-Arabinose</td>
			<td style="width:127px;">Sigma: http://www.sigmaaldrich.com</td>
			<td style="width:216px;">A3256-25G</td>
			<td></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:315px;">Long oligos</td>
			<td style="width:127px;">IDT: https://www.idtdna.com; Gene link: https://www.genelink.com</td>
			<td style="width:216px;">IDT Ultramers or Gene Link long oligos</td>
			<td>PTO and Phosphosphate available as custom modifications. PAGE purification strongly recommended for long oligos.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">MinElute PCR purification kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">28004</td>
			<td>Minimum elution PCR purifications kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">QIAPrep Spin Mini Prep kit</td>
			<td style="width:127px;">Qiagen: http://www.qiagen.com&#160;</td>
			<td align="right" style="width:216px;">27104</td>
			<td>Silica column or similar matrix kits are preferred.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:315px;">Restriction enzymes</td>
			<td style="width:127px;">NEB: https://www.neb.com</td>
			<td style="width:216px;">DpnI: R0176L</td>
			<td>See NEB website for a list of RE.</td>
		</tr>
	</tbody>
</table>

subcloning plus insertion (spi) - a novel recombineering method for the rapid construction of gene targeting vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Watch this Scientific Journal Video about Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors at JoVE.com

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

استنساخ فرعي زائد الإدراج (SPI) - رواية الطريقة Recombineering عن البناء السريع للجين استهداف ناقلات

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

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Biology

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

16.4K Views.  University of Leicester. Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term ‘subcloning plus insertion’, which can be used to generate custom gene targeting vectors rapidly.

Video: Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

سريع سرعة عالية طريقة لرسم الخرائط Ribonucleoproteins (RNPs) على مرنا ما قبل الإنسان

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

مرحبا - C : طريقة لدراسة الهندسة المعمارية ثلاثي الأبعاد للخريطة جينوم.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

لإنتاج C. ايليجانس الجينات المحورة عبر Recombineering مع galK إختيار ماركر

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

طريقة مبتكرة لثقافة الاستقطاب وتحفيز من explants الإنسان مخاطية الأمعاء

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

Recombineering مثلي التركيبات التوحد في<em&gt; ذبابة الفاكهة</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

رواية<em&gt; خارج الحي</em&gt; الطريقة الثقافة لالجنينية القلب

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell populations.&#160;These cultures contain the cells of interest, but are also contaminated with undifferentiated ESCs, non-neural derivatives and other neuronal subtypes.&#160; This limits their use in in vitro and in vivo applications, such as in vitro modeling for drug discovery or cell replacement therapy. To help overcome this, reporter cell lines, which offer a means to visualize, track and isolate cells of interest, can be engineered.&#160;However, to achieve this in human embryonic stem cells via conventional homologous recombination is extremely inefficient.&#160;This protocol describes targeting of the Pituitary homeobox 3 (PITX3) locus in human embryonic stem cells using custom designed zinc-finger nucleases, which introduce site-specific double-strand DNA breaks, together with a PITX3-EGFP-specific DNA donor vector. Following the generation of the PITX3 reporter cell line, it can then be differentiated using published protocols for use in studies such as in vitro Parkinson&#8217;s disease modeling or cell replacement therapy.

Reporter cell lines offer a means to visualize, track and isolate cells of interest from heterogeneous populations.&#160;However, gene-targeting using conventional homologous recombination in human embryonic stem cells is extremely inefficient. Herein, we describe targeting CNS midbrain specific transcription factor PITX3 locus with EGFP using zinc-finger nuclease enhanced homologous recombination.

الزنك الاصبع نوكلياز المحسن استهداف الجينات في الخلايا الجذعية الجنينية البشرية

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

عزل الترشيح من الأحماض النووية: وبسيطة وسريعة استخراج الحمض النووي الطريقة

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Rapid, Scalable Method for the Isolation, Functional Study, and Analysis of Cell-derived Extracellular Matrix

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and pathological processes. However, the isolation of ECM, from tissues or cell culture, is complicated by the insoluble and cross-linked nature of the assembled ECM and by the potential contamination of ECM extracts with cell surface and intracellular proteins. Here, we describe a method for use with cultured cells that is rapid and reliably removes cells to isolate a cell-derived ECM for downstream experimentation.
Through use of this method, the isolated ECM and its components can be visualized by in situ immunofluorescence microscopy. The dynamics of specific ECM proteins can be tracked by tracing the deposition of a tagged protein using fluorescence microscopy, both before and after the removal of cells. Alternatively, the isolated ECM can be extracted for biochemical analysis, such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. At larger scales, a full proteomics analysis of the isolated ECM by mass spectrometry can be conducted. By conducting ECM isolation under sterile conditions, sterile ECM layers can be obtained for functional or phenotypic studies with any cell of interest. The method can be applied to any adherent cell type, is relatively easy to perform, and can be linked to a wide repertoire of experimental designs.

The extracellular matrix plays a major role in defining the microenvironment of cells and in modulating cell behavior and phenotype. We describe a rapid method for the isolation of cell-derived extracellular matrix, which can be adapted to different scales for microscopic, biochemical, proteomic, or functional studies.

والسريعة، طريقة قابلة للعزل، دراسة وظيفية، وتحليل المصفوفة خارج الخلية المشتقة من خلية

The extracellular matrix (ECM) is recognized as a diverse, dynamic, and complex environment that is involved in multiple cell-physiological and ...

Protocols for Testing the Toxicity of Novel Insecticidal Chemistries to Mosquitoes

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such as Zika, dengue and malaria. Assays for rapid, high-throughput analyses of unformulated novel chemistries against mosquito larvae and adults are presented. We describe protocols for single point-dose and dose response assays to evaluate the toxicity of small molecule chemistries to the Aedes aegypti vector of Zika, dengue and yellow fever,&#160;the malaria vector, Anopheles gambiae and the northern house mosquito, Culex quinquefasciatus, on contact and via ingestion. As an example, we evaluated the toxicity of amitriptyline, a small molecule antagonist of G protein-coupled receptors, via larval, adult topical and adult blood-feeding assay. The protocols provide a starting point to investigate insecticide potential. Results are discussed in the context of additional experiments to explore product applications and mechanisms for delivery.

Protocols are described for assessing the toxicity of chemistries to immature and adult mosquitoes for development as new classes of larvicides, adulticides and endectocides. The protocols enable high-throughput testing of multiple chemistries at single-point dose and subsequent evaluation via dose response assay to determine toxicity on contact or via ingestion.

بروتوكولات لاختبار السمية لرواية كيمياء مبيدات الحشرات للبعوض

New classes of insecticides with novel modes of action are needed to control insecticide resistant populations of mosquitoes that transmit diseases such ...