Name: a fast air-dry dropping chromosome preparation method suitable for fish in plants
Uploaded: 2015-12-16T14:00:00.000+00:00
Duration: 9 min 8 s
Description: Watch this Scientific Journal Video about A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants at JoVE.com

We gratefully thank the DFG for financial support (HO 1779/21-1) as well as Katrin Kumke and Dr. Veit Schubert (IPK, Gatersleben) for technical advice.

1. إعداد كروموسوم <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> إنبات البذور والتثبيت من النصائح الجذر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تنبت 10-20 بذور الشعير على طبقتين من ورق الترشيح رطبة في طبق بيتري في ظل ظروف مظلمة لمدة 2 أيام في 22-24 درجة مئوية. قطع جذور قوية مع طول 1-2 سم من البذور باستخدام شفرة حلاقة. </li><li style=";text-align:right;direction:rtl"> تحضير الماء المثلج عن طريق وضع زجاجة 500 مل يحتوي على ماء الصنبور البارد في سحقت الماء المثلج. تهوية الماء المثلج وتغمر نصائح الجذرية لمدة 20 ساعة لزيادة وتيرة الخلايا الطورية. </li><li style=";text-align:right;direction:rtl"> نقل جذور من المياه إلى 50 مل من الايثانول: حامض الخليك (3: 1) تثبيتي لإصلاح لهم في RT لمدة 2 أيام. متجر جذور في الإيثانول الطازجة: حمض الخليك (3: 1) تثبيتي في 4 درجات مئوية حتى استخدام ما يصل إلى سنة. </li></ol></li><li style=";text-align:right;direction:rtl"> غسل وانزيم العلاج <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> غسل 10-20 الجذور مع 30 مل من الجليد الباردة ماء الصنبور لمدة 5 دقائق مرتين باستخدام كوب 50 مل دورق. استخدام المجهر مجهر. نقل جذور واحدا تلو الآخر إلى30 مل 0،01 M عازلة سيترات (0.01 حمض الستريك M + M 0.01 سيترات الصوديوم، ودرجة الحموضة 4.8) باستخدام ملقط، ويغسل عن طريق هز كوب زجاجي لمدة 5 دقائق مرتين. وضع الجذور على الورق مرشح لإزالة السائل تماما وقطع الأنسجة غير بارضي غير مرغوب فيها باستخدام شفرة حلاقة. </li><li style=";text-align:right;direction:rtl"> احتضان ما يصل الى 20 نصائح الجذرية في 1 مل خليط الانزيم عند 37 درجة مئوية لمدة 50 دقيقة لتليين الأنسجة النباتية (الجدول 1) في الزجاج ووتش. يحتوي على خليط انزيم 0.7٪ R10 السيلولوز، 0.7٪ السيلولوز، 1٪ pectolyase و 1٪ cytohelicase مخففة في 0.01 M عازلة سيترات. تخزين خليط الانزيم في -20 ° C وإعادة استخدامها ما يصل الى خمس مرات. </li><li style=";text-align:right;direction:rtl"> إزالة الانزيم من قبل pipetting وغسل نصائح الجذر على الجليد مع 5 مل 0.01 M عازلة سيترات مرتين ليحل محل الانزيم المتبقية. </li></ol></li><li style=";text-align:right;direction:rtl"> النقع الجذر <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> نصائح الجذر غسل مع 1 مل 96٪ من الإيثانول مرتين بعناية في نفس الزجاج ووتش. استبدال الإيثانول مع الطازجة تثبيتي (75٪ حامض الخليك: 25٪ من الإيثانول). استخدام 10-15تثبيتي ميكرولتر في قمة الجذر. </li><li style=";text-align:right;direction:rtl"> نصائح نقل الجذر جنبا إلى جنب مع تثبيتي في أنبوب 2 مل وتتفكك الخلايا الإنشائية الجذر مع إبرة تشريح أو ملقط. الاستفادة من أنبوب 20 مرات لإعادة تعليق الخلايا للحصول على تعليق خلية. تخزين تعليق خلية في -20 ° C تصل إلى شهرين. </li></ol></li><li style=";text-align:right;direction:rtl"> إسقاط تعليق الخلية <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ضع 2-3 طبقات من المناديل الورقية غارقة في المياه على طبق ساخن عند 50 درجة مئوية. تزج الشرائح المجهرية في مياه الحنفية المثلج في الثلاجة لمدة 30 دقيقة. والشرائح مكان على أعلى من المناديل الورقية الرطبة. </li><li style=";text-align:right;direction:rtl"> ماصة 7-10 ميكرولتر من تعليق خلية وأسقطه من مسافة 20 سم على الشريحة تبريد توضع على طبق ساخن. ماصة 10 ميكرولتر من الخل حامض خليط الإيثانول في نفس المكان تعليق الخلية على الشريحة والحفاظ على الشريحة على طبق ساخن لمدة 2 دقيقة إضافية. وضع الشريحة على طبق ساخن بدون النسيج الرطب واتركها تجف لمدة 1 دقيقة. </li></ol></li><li style=";text-align:right;direction:rtl"> مراقبة الجودة وstorag(ه) من الشرائح <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحقق الشرائح باستخدام المجهر المرحلة على النقيض من لمراقبة جودة انتشار كروموسوم. استخدام الشرائح إما في نفس اليوم أو مخزن عن طريق غمر في 96٪ من الإيثانول في وعاء Coplin في -20 ° C. </li></ol></li><li style=";text-align:right;direction:rtl"> المعالجة من قبل الشرائح FISH. تتم جميع الخطوات من في RT <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مكان الشرائح في جرة Coplin تحتوي على 50 مل من 2X SSC (20x وSSC يحتوي على 3 M كلوريد الصوديوم و 300 ملي سترات الصوديوم) لمدة 5 دقائق. باستخدام ملقط، والشرائح نقل إلى جرة Coplin تحتوي على 50 مل من حمض الخليك 45٪ ل3-10 دقيقة. </li><li style=";text-align:right;direction:rtl"> نقل الشرائح إلى جرة Coplin تحتوي على 50 مل من 2X SSC لمدة 10 دقيقة. الشرائح نقل إلى جرة Coplin تحتوي على 50 مل من 4٪ الفورمالديهايد (في 2X SSC) وتزج الشرائح لمدة 10 دقيقة لإصلاح الصبغيات. </li><li style=";text-align:right;direction:rtl"> إزالة الفورمالديهايد قبل الشطف الشرائح 3 مرات لمدة 4 دقائق لكل منهما، في جرة Coplin تحتوي على 50 مل 2X SSC. يذوى الشرائح في جرة Coplin لمدة 2 دقيقة في سلسلة من 70٪، 90٪ و 100٪ من الإيثانول، على التوالي والشرائح الجافة في رأسيموضع. </li></ol></li></ol> 2. نيون في الموقع التهجين (FISH) <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> لكل شريحة، يعد حل التهجين 20 ميكرولتر في المجموع باستخدام 10 ميكرولتر من الفورماميد منزوع الأيونات، 5 ميكرولتر من العازلة 4X التهجين (200 العازلة ميكرولتر يحتوي على 80 ميكرولتر 20X SSC، 8 ميكرولتر 1 M تريس، حمض الهيدروكلوريك درجة الحموضة 8.0، 1.6 ميكرولتر 0.5 M EDTA، 11.2 ميكرولتر 10 ميكروغرام / ميكرولتر الحيوانات المنوية السلمون و99،2 ميكرولتر خالية من الدناز الماء)، و 3 ميكرولتر من التحقيق و2 ميكرولتر من المياه خالية من الدناز. </li><li style=";text-align:right;direction:rtl"> إضافة 20 ميكرولتر من محلول التهجين لكل شريحة والغطاء مع 24 × 32 مم زلة تغطية وإلقاء القبض على زلة غطاء مع الاسمنت والمطاط. تفسد الشرائح مع تحقيقات في وقت واحد في 80 درجة مئوية لمدة 2 دقيقة على طبق ساخن. </li><li style=";text-align:right;direction:rtl"> نقل الشرائح إلى غرفة رطبة واحتضان الشرائح في 37 ° CO / N ضوء تجنب. إزالة زلات غطاء من قبل الشطف الشرائح في جرة Coplin مع 2X SSC. مكان الشرائح في جرة Coplin تحتوي على 55-60 ° C 2X SSC واحتضان لمدة 20 دقيقة. </li><li style=";text-align:right;direction:rtl"> ضع الشرائح إلى 2x SSC في جرة Coplin لمدة 2 دقيقة في RT. يذوى الشرائح في جرة Coplin لمدة 2 دقيقة في سلسلة من 70٪، 90٪ و 100٪ من الإيثانول، على التوالي. </li><li style=";text-align:right;direction:rtl"> الهواء الجاف الشرائح ومباين مع 1 ميكروغرام / مل 4، 6-diamidino-2-phenylindoline (دابي) في antifade المتوسطة المتزايدة، وتجنب الضوء المكثف. </li></ol> 3. تحليل مجهري والتخزين <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تحليل الشرائح باستخدام المجهر epifluorescence. اختيار مرشح يعتمد على تألقي تستخدم لوضع العلامات التحقيق. إذا لزم الأمر، والشرائح تخزينها في 4 درجات مئوية تحت الظروف المظلمة تصل الى عام. </li></ol>

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The authors have nothing to disclose.

وقد تم تنفيذ التجربة إعداد كروموسوم من استخدام جذور الشباب من الحبوب تعود لعائلة الأعشاب (النجيلية). جميع الأنواع تحليل لها 14 الإنقسامية طويلة نسبيا الكروموسومات الطورية (11-15 ميكرون) في مجموعة الجينوم مضاعفا وتنتمي إلى الأنواع واسع الجينوم (5،1-7،9 GBP). <p class="jove_content" sty...

مضان التهجين في الموقع (FISH) في هو أداة فعالة لرسم خرائط المادي للتسلسل نسخة واحدة وعالية على مستوى الكروموسومات. شرط أساسي هو إعداد ينتشر كروموسوم جودة عالية. ليس هناك كروموسوم العام إعداد بروتوكول التي من شأنها أن تكون مناسبة أيضا للخلايا الحيوانية والنباتية. إعداد الكروموسومات المصنع هو تحديا من نوع خاص بسبب جدار الخلية جامدة ومختلف الاتساق السيتوبلازم داخل الأنواع المختلفة. إحدى الطرق الملائمة لإعداد الصبغيات النباتية هو ما يسمى تقنية انخفاض المعروف أيضا باسم تقنية الانتشار قطرة وتجفيف الهواء تقنية 1،2. وقدم هذه الطريقة لأول مرة في عام 1958 من قبل Rothfels وSiminovitch لفي المختبر خلايا الثدييات نمت 3. في وقت لاحق مارتن وآخرون. (4) وكاتو وآخرون. 5 تكييف هذه الطريقة للنباتات. وفي الآونة الأخيرة، وهي طريقة تسمى &quot;SteamDrop &amp;# 39؛ وقد وضعت والتي تستخدم البخار المياه لإعداد الكروموسومات غير متداخلة 6. على الرغم من أن لوحظ تأثير إيجابي من ارتفاع نسبة الرطوبة في وقت سابق من 7، &#39;SteamDrop&#39; يسلم سير العمل الخاضعة للرقابة ذات جودة عالية التحضيرات كروموسوم 6. يؤدي العلاج بالبخار تمتد من الكروموسومات ربما تتصل بعض التعديلات من البروتينات الكروموسومات. نوعية الناتج ينتشر الطورية عالية جدا، على الرغم من الاحتفاظ بعدد كاف من الطورية ينتشر كاملة للتجارب FISH اللاحقة يطالب الخبرة التقنية. هنا نقدم بروتوكول لإعداد الكروموسومات الإنقسامية الحبوب مناسبة للكشف FISH من واحد ونسخة عالية تحقيقات 5،8. هذا الأسلوب هو البديل الأفضل للطريقة إسقاط الهواء الجاف وصفه كاتو 9 تتم تحت الرطوبة النسبية من 50٪ -55٪ (الشكل 1). ويتألف هذا البروتوكول عددا أقلغسل خطوات صنع تطبيقه سهلة وفعالة وقابلة للتكرار. باستخدام هذا البروتوكول حصلنا على هوامش كروموسوم ذات جودة عالية ونتائج FISH لفولغاري شعير، H. bulbosum، H. marinum، H. murinum، H. pubiflorum والشيلم cereale.

<table><tbody><tr><td>Hot Plate</td><td>MEDAX GmbH</td><td>12603</td><td></td></tr><tr><td>Cellulase R10</td><td>Duchefa</td><td>C8001</td><td></td></tr><tr><td>Cellulase&amp;nbsp;</td><td>CalBioChem</td><td>219466</td><td></td></tr><tr><td>Pectolyase</td><td>Sigma</td><td>P3026</td><td></td></tr><tr><td>Cytohelicase</td><td>Sigma</td><td>C8274</td><td></td></tr><tr><td>Texas Red-12-dUTP</td><td>Invitrogen</td><td>C3176</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluor488-5-dUTP</td><td>Invitrogen</td><td>C11397</td><td>direct fluorochrome&amp;nbsp;</td></tr><tr><td>Fluorecsence microscope</td><td>Olympus BX61</td><td>BX61</td><td></td></tr><tr><td>CCD camera</td><td>Orca ER, Hamamatsu</td><td>C10600</td><td></td></tr><tr><td>4&amp;rsquo;,6-diamidino-2-phenylindole (DAPI)&amp;nbsp;</td><td>Vector Laboratories</td><td>H-1200</td><td>fluorecsent dye</td></tr></tbody></table>

a fast air-dry dropping chromosome preparation method suitable for fish in plants

إعداد ينتشر الكروموسومات هي شرط أساسي لنجاح أداء مضان في الموقع التهجين (FISH). إعداد جودة عالية ينتشر كروموسوم مصنع يمثل تحديا بسبب جدار الخلية جامدة. إحدى الطرق المعتمدة لإعداد الصبغيات النباتية هو ما يسمى إعداد قطرة، المعروف أيضا باسم نشر قطرة أو تقنية تجفيف الهواء. هنا، نقدم بروتوكول لإعداد سريع من كروموسوم الإنقسامية ينتشر مناسبة للكشف FISH تحقيقات DNA نسخة واحدة والعالية. هذا الأسلوب هو البديل الأفضل للطريقة قطرة الهواء الجاف المنفذة بموجب عقد الرطوبة النسبية من 50٪ -55٪. ويتألف هذا البروتوكول انخفاض عدد غسل خطوات صنع تطبيقه سهلة وفعالة وقابلة للتكرار. فوائد واضحة لهذا النهج بشكل جيد الانتشار، الكروموسومات الطورية التالفة والعديد من العاملين كشرط مسبق مثالية لتحليل FISH ناجحة. باستخدام هذا البروتوكول حصلنا عالية الجودة الكرومosome ينتشر والنتائج FISH استنساخه لفولغاري شعير، H. bulbosum، H. marinum، H. murinum، H. pubiflorum والشيلم cereale.

تم إعداد الشرائح المجهرية مع هوامش الطورية الإنقسامية قبل إسقاط الصبغي سريع طريقة التحضير الهواء الجاف هو موضح أعلاه (ملحق الشكل 1). وأجري تحليل FISH من استخدام كليهما، تسلسل المتكررة ونسخة واحدة. وقد تم الحصول على الصور عن طريق المجهر epifluorescence مع مجموعة من الم...

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A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

سريع الهواء الجاف اسقاط كروموسوم طريقة التحضير مناسبة لFISH في النباتات

Preparation of chromosome spreads is a prerequisite for the successful performance of fluorescence in situ hybridization (FISH). Preparation of high ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.2K Views.  Leibniz Institute of Plant Genetics and Crop Plant Research (IPK). A protocol is described for the preparation of high-quality mitotic plant chromosome spreads by a fast air-dry dropping method suitable for the FISH detection of single and high copy DNA probes.

Video: A Fast Air-dry Dropping Chromosome Preparation Method Suitable for FISH in Plants

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

التنميط الحمض النووي توقيت النسخ المتماثل باستخدام الزرد كنظام نموذجي في فيفو

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Genetics

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

التلاعب الصيغة الصبغية من الزرد الأجنة مع الحرارة صدمة 2 العلاج

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, ...

Developmental Biology

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

فلوري<em&gt; في الموقع</em&gt; التهجين على الكروموسومات الميتوزى من البعوض

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Immunology and Infection

Preparation of Meiotic Chromosome Spreads from Zebrafish Spermatocytes

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding the chromosome events that take place during meiotic prophase, including the pairing, synapsis, and recombination events that ensure proper chromosome segregation. While the mouse has been an important model for understanding the molecular mechanisms underlying these processes, not all meiotic events in this system are analogous to human meiosis. We recently demonstrated the exciting potential of the zebrafish as a model of human spermatogenesis. Here we describe, in detail, our methods to visualize meiotic chromosomes and associated proteins in chromosome spread preparations. These preparations have the advantage of allowing high resolution analysis of chromosome structures. First, we describe the procedure for dissecting testes from adult zebrafish, followed by cell dissociation, lysis, and spreading of the chromosomes. Next, we describe the procedure for detecting the localization of meiotic chromosome proteins, by immunofluorescence detection, and nucleic acid sequences, by fluorescence in situ hybridization (FISH). These techniques comprise a useful set of tools for the cytological analysis of meiotic chromatin architecture in the zebrafish system. Researchers in the zebrafish community should be able to quickly master these techniques and incorporate them into their standard analyses of reproductive function.

Nuclear surface spreads are an indispensable tool for studying chromosome events during meiosis. Here we demonstrate a method to prepare and visualize meiotic chromosomes during prophase I from zebrafish spermatocytes.

إعداد ينتشر كروموسوم Meiotic من الحيوانات المنوية زيبرافيش

Meiosis is the key cellular process required to create haploid gametes for sexual reproduction. Model organisms have been instrumental in understanding ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

التعبير المشارك من البروتينات الانصهار الفلورسنت تشيميريك متعددة بطريقة فعالة في النباتات

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Chemistry

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

2D و 3D كروموسوم اللوحة في البعوض الملاريا

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

أسلوب الإنتاجية العالية للحصول على الحيوانات المنوية الحفظ بالتبريد واسماك الزرد في المختبر التسميد

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Making Gynogenetic Diploid Zebrafish by Early Pressure

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2 described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3.

This is a method for generating gynogenetic diploid zebrafish embryos (embryos whose only genetic contribution comes from the mother) by blocking the second meiotic division immediately after fertilization with ultraviolet light-inactivated sperm. EP embryos are not fully homozygous due to recombination during the first meiotic division, however they are homozygous at all loci that have not been separated from their centromere by recombination.

جعل Gynogenetic اسماك الزرد ضعفاني بواسطة الضغط المبكر

Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from ...

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.

The material here describes a method developed to preserve the three-dimensional chromatin structure of testicular germ cells. This has been termed the three-dimensional (3D) slide method. This method improves sensitivity for detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH).

طريقة إعداد الشرائح للحفاظ على هندسة الكروماتين ثلاثية الأبعاد للخلايا الجرثومية الخصية

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

سريع الهواء الجاف اسقاط كروموسوم طريقة التحضير مناسبة لFISH في النباتات

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