وأيد هذا العمل من قبل مؤسسة العلوم الوطنية وIOS IOS 0844948 1024811

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A., Bradham, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pantropic+retroviruses+as+a+transduction+tool+for+sea+urchin+embryos.">Pantropic retroviruses as a transduction tool for sea urchin embryos.</a> Proc. Natl. Acad. Sci. U. S. A. 109, 5334-5339 (2012).</li></ol>

أي تضارب في المصالح أعلنت من قبل المؤلفين.

هناك نوعان من الخطوات الحاسمة التي من الصعب بالنسبة للمستخدمين المبتدئين لهذه التقنية ولكن لا غنى عنها لخلق أجنة morphant بنجاح. الأول هو اختيار البويضات السليمة التي من شأنها أن تنضج وتسميد بشكل صحيح. نسبة التطور الطبيعي في ثقافة تعتمد على الموسم، وصحة الحيوان، وعدد ال...

نجم البحر، Patiria miniata، (المعروف باسم نجم الخفافيش) في الظهور كنظام نموذج للاهتمام وتنوعا لمجموعة متنوعة من الخلايا 1- 3، 4،5 التنموية، التطورية 6- 8، والدراسات البيئية 9- 11. الكبار P. وتوزع miniata على طول ساحل المحيط الهادئ من سيتكا، ألاسكا إلى باجا كاليفورنيا 12 ويتم الاحتفاظ بسهولة في الأحواض المائية البحرية. البويضات التي يمكن الحصول عليها على مدار السنة وكل أنثى يمكن إلقاء عشرات الآلاف من البيض. ونضجت بسهولة البويضات المخصبة وخارجيا 13. الأجنة الناتجة شفافة مما يسمح للمراقبة سهلة؛ أن تتطور بشكل متزامن، وتحتاج إلى مياه البحر الوحيد للتنمية. التجمع الجينوم كله وtranscriptomes متعددة متاحة للP. أيضا miniata ( <a href="http://www.echinobase.org/Echinobase/">Echinobase.org</a> ). هذه المزايا تجعل يجعلها مثالية لمجموعة واسعة من البحوثوأغراض التدريس. في السنوات الأخيرة، P. أصبح miniata نظام نموذجي لشبكة تنظيمية الجينات التنموي يحلل 14- 16. والهدف من هذه الدراسات هو تحديد مجاملة كاملة من الجينات التنظيمية وتحديد شبكة من تفاعلاتها. الكثير من هذا العمل ينطوي على التعبير الجيني من خلال إقلاق إدخال أليغنوكليوتيد] العقاقير أو في المختبر توليفها من mRNAs. بالإضافة إلى ذلك، يتم استخدام التحليلات التنظيمية رابطة الدول المستقلة لتوصيف وظيفة الحمض النووي التنظيمي 15. هذه التحليلات تتطلب إدخال الكواشف اضطراب و / أو مراسل الحمض النووي يبني في الأجنة. وعلاوة على ذلك، لوصف آثار المصب من هذه الاضطرابات، لا بد من مقايسة العديد من الأجنة لإجراء تغييرات في التعبير الجيني من الأهداف المحتملة. تقنيات Microinjection من عدة مئات من بيضات ملقحة هي المركزية لهذا العمل. شوكيات الجلد، بما في ذلك P. miniata </م&gt;، يتطلب عدة أشهر لتصل إلى مرحلة النضج الجنسي. وبسبب هذا، فهو عموما ليس العملي لتطوير وصيانة خطوط المعدلة وراثيا من هذه الحيوانات للتجريب. لذا، تربية البالغين المعدلة وراثيا لا يمكن بكفاءة خلق الأجنة تعديل. بدلا من ذلك، يجب أن يحدث الاضطراب من جديد من خلال حقن مكروي. يقدم حقن مكروي فرصة لتعديل الأجنة مع الكواشف التي ليست خلية منفذة. يصف بروتوكول التالية طريقة لإدخال DNA، مرنا، استشفاف خلية، وأليغنوكليوتيد] morpholino العقاقير إلى مئات من البويضات المخصبة في واحدة 2-3 ساعة يجلس من خلال حقن مكروي. هذا وتنتج مادة كافية لمجموعة متنوعة من التجارب المصب بما في ذلك، ولكن ليس على سبيل الحصر، QPCR، الموقع التهجين، RNA تسلسل، والنشاف الغربي في.

<table><tbody><tr><td>1-Methyladenine</td><td>Acros Organics (Fisher Scientific)</td><td>AC20131-1000</td><td></td></tr><tr><td>190 micron nitex nylon filter</td><td>Small Parts (originally Sefar)</td><td>CMN-0185-C/5PK-05</td><td></td></tr><tr><td>100 micron nitex nylon filter</td><td>Small Parts (originally Sefar)</td><td>CMN-0105-C/5PK-05</td><td></td></tr><tr><td>Polystyrene Petri dishes, 60 mm x 15 mm</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Capillary tubing</td><td>FHC, Inc</td><td>30-30-0</td><td>For pulling microinjection needles</td></tr><tr><td>Model P-97 Needle Puller</td><td>Sutter Instruments</td><td>P-97</td><td></td></tr><tr><td>Dextran, Rhodamine Green</td><td>Life Technologies</td><td>D7163</td><td>If injecting a GFP expression reporter, it is helpful to substitute Texas Red dextran as an injection tracer</td></tr><tr><td>Instant Ocean Sea Salt</td><td>Doctors Foster and Smith</td><td>CD-116528</td><td>Also available in many pet stores</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>5242 956.003</td><td></td></tr></tbody></table>

a method for microinjection of patiria minata zygotes

كانت شوكيات الجلد لفترة طويلة نظام نموذجي المفضل للدراسات الاستنساخ والتنمية، ومؤخرا لدراسة تنظيم الجينات وتطور العمليات التنموية. نجم البحر، Patiria miniata، تكتسب انتشار كنظام نموذج لهذه الأنواع من الدراسات التي أجريت سابقا على وجه الحصر تقريبا في قنافذ البحر، Strongylocentrotus purpuratus وLytechinus variegatus. ميزة هذه الأنظمة النموذج هو سهولة إنتاج أجنة تعديل فيها هو جين معين صعودا أو downregulated، ووضع العلامات مجموعة من الخلايا، أو إدخال الجين المراسل. وهناك طريقة حقن مكروي واحد قادر على خلق مجموعة واسعة من هذه الأجنة تعديل. هنا، فإننا نقدم وسيلة للحصول على الأمشاج من P. miniata، وإنتاج بيضات ملقحة، وإدخال الكواشف مشوشة عبر حقن مكروي. يتم عزل الأجنة morphant صحية في وقت لاحق للدراسات الكمية والنوعية للجنرال الكتريكوظيفة الشرق الأدنى. توافر الجينوم وTranscriptome على البيانات لهذا الكائن الحي ازدادت أنواع الدراسات التي يتم تنفيذها وسهولة تنفيذها.

الهدف من هذا البروتوكول هو تقديم الكواشف إلى الأجنة. نحن إثبات فعالية البروتوكول عن طريق حقن مراسل بناء الحمض النووي الذي يدفع التعبير عن بروتين الفلورية الخضراء (GFP). حقن الأجنة تعبر عن GFP في بقع نسيلي (الشكل 4A-B) والحمض النووي خلال الانقسام يتضمن المبكر. العد?...

Watch this Scientific Journal Video about A Method for Microinjection of Patiria minata Zygotes at JoVE.com

A Method for Microinjection of Patiria minata Zygotes

الأساليب التي تنتج أجنة morphant ضرورية لدراسة آليات تنموية والشبكات التنظيمية الجينات. نجم البحر Patiria miniata هو نظام نموذجي الناشئة عن هذه الدراسات. هنا نقدم بروتوكول للحصول على الأمشاج، وتنتج ثقافات الأجنة، وحقن مكروي السريع للبيضات ملقحة من هذا النوع.

منهج في Microinjection من<em&gt; Patiria minata</em&gt; بيضات ملقحة

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

a-method-for-microinjection-of-patiria-minata-zygotes

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

A Method for Microinjection of Patiria minata Zygotes

10.9K Views.  Carnegie Mellon University.  الأساليب التي تنتج أجنة morphant ضرورية لدراسة آليات تنموية والشبكات التنظيمية الجينات. نجم البحر Patiria miniata هو نظام نموذجي الناشئة عن هذه الدراسات. هنا نقدم بروتوكول للحصول على الأمشاج، وتنتج ثقافات الأجنة، وحقن مكروي السريع للبيضات ملقح...

Video: A Method for Microinjection of Patiria minata Zygotes

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

حشوية حقن مكروي في الثروة الحيوانية بيضات ملقحة بمساعدة الليزر

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Genetics

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

تحليل التنمية الكلى الزرد مع الوقت الفاصل بين التصوير باستخدام مجهر تشريح مجهزة لباجتزاء البصري

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Developmental Biology

Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

Microinjection is a well-established and effective method for introducing foreign substances into fertilized zebrafish embryos. Here, we demonstrate a robust microinjection technique for performing mRNA overexpression, and morpholino oligonucleotide gene knockdown studies in zebrafish.

Microinjection من مرنا والعقاقير [أليغنوكليوتيد] Morpholino اسماك الزرد في الأجنة.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinjection الجنين اسماك الزرد إلى تحليل وظائف الجينات

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

أجهزة ميكروستروكتوريد Microinjection الأمثل وتصوير يرقات الزرد

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Microinjection Method for Anopheles gambiae Embryos

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA fragments encoding genes of interest as well as favorable traits into the insect germline in a stable and heritable manner. The resulting transgenic strains can be studied for phenotypic changes resulting from the expression of the integrated DNA to answer basic questions or used in practical applications. Although the technology is straightforward, it requires of the investigator patience and practice to achieve a level of skill that maximizes efficiency. Shown here is a method for microinjection of embryos of the African malaria mosquito, Anopheles gambiae. The objective is to deliver by microinjection exogenous DNA to the embryo so that it can be taken up in the developing germline (pole) cells. Expression from the injected DNA of transposases, integrases, recombinases, or other nucleases (for example CRISPR-associated proteins, Cas) can trigger events that lead to its covalent insertion into chromosomes. Transgenic An. gambiae generated from these technologies have been used for basic studies of immune system components, genes involved in blood-feeding, and elements of the olfactory system. In addition, these techniques have been used to produce An. gambiae strains with traits that may help control the transmission of malaria parasites.

Microinjection Method for Anopheles gambiae Embryos

Microinjection techniques are essential to introduce exogenous genes into the genomes of mosquitoes. This protocol explains a method used by the James laboratory to microinject DNA constructs into Anopheles gambiae embryos to generate transformed mosquitoes.

طريقة الميكروبيكشن للأجنة الغامبية Anopheles

Embryo microinjection techniques are essential for many molecular and genetic studies of insect species. They provide a means to introduce exogenous DNA ...

Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily produced by electroporation of CRISPR factors (and single-stranded oligo DNA donors) into the zygote. In contrast, gene cassette (&#62;1 kb) knock-in and floxed mice are mainly generated by microinjection of CRISPR factors and double-stranded DNA donors into zygotes. Genome editing technologies have also increased the flexibility of genetically modified mice production. It is now possible to introduce the intended mutations in the target genomic regions in a number of beneficial inbred mouse strains. Our team has produced over 200 gene cassette knock-in mouse lines, and over 110 floxed mouse lines by zygote microinjection of CRISPR-Cas9 following requests from several countries, including Japan. Some of these genome editing used BALB/c, C3H/HeJ, and C57BL/6N inbred strains, however most used C57BL/6J. Unlike the electroporation method, genome editing by zygote microinjection in various inbred strains of mice is not that easy. However, gene cassette knock-in and floxed mice on single inbred genetic backgrounds are as critical as genetic humanized, fluorescent reporter, and conditional knockout mouse models. Therefore, this article presents the protocol for the zygote microinjection of CRISPR factors and double-stranded DNA donors in C57BL/6J mice for generating gene cassette knock-in and floxed mice. This article exclusively focuses on nuclear injection rather than cytoplasmic injection. In addition to zygote microinjection, we outline the timeline for the production process and peripheral techniques such as induction of superovulation and embryo transfer.

The present protocol describes zygote microinjection of CRISPR-Cas9 and donor DNA to efficiently produce gene cassette knock-in and floxed mice.

الحقن المجهري للزيجوت لإنشاء أليلات كاسيت الجينات وأليلات فلوكس في الفئران

CRISPR-Cas technology has enabled the rapid and effortless generation of genetically modified mice. Specifically, mice and point mutant mice are readily ...

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (dsRNA) into the gonads to obtain non-rolling F1 progeny. Unlike C. elegans, but like most other nematodes, P. pacificus PS312 is not receptive to systemic RNAi via feeding and soaking and therefore dsRNA must be administered by microinjection into the syncitial gonads. In this current study, we hope to describe the microinjection process needed to transform a Ppa-egl-4 promoter::GFP fusion reporter and knockdown a dominant roller prl-1 (tu92) mutant in a visually informative protocol.

RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

In model organisms, transgenesis can manipulate gene functions while RNAi can knockdown specific mRNA transcripts 1-2. This protocol aims to illustrate the techniques needed to introduce stably transmitted DNA and transient double stranded RNA into the necromenic nematode Pristionchus pacificus for studies in evolutionary, developmental, and behavioral biology.

وساطة رني ضربة قاضية بواسطة الجينات وTransgenesis Microinjection في نيماتودا Necromenic Pristionchus pacificus</em

Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

عالية الإنتاجية إبر دقيقة جدا لقنفذ البحر بيضات ملقحة

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Zebrafish Microinjection Techniques

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage embryos. Using this technique, solutions containing genetic material or knockdown constructs are delivered into the blastomeres: the embryonic cells sitting atop the yolk of the newly fertilized egg. Delivery into the cytoplasm is achieved either through direct injection into the blastomere, or via natural cytoplasmic movements that occur after a solution is injected into the yolk. Successful genetic manipulations are usually followed by quantification of embryonic phenotypes in order to elucidate the genetic mechanisms of development.
This video will provide an introduction to carrying out microinjections in zebrafish embryos. The discussion begins with a review of the essential tools for the technique, including the injection apparatus and the microinjector, which controls fluid movement with pressure pulses of air. Next, important preparatory steps are demonstrated, such as the pouring of agar plates to stabilize embryos during injection and calibration of the microinjection apparatus. The injection procedure is then presented along with tips on when and where injections should be performed. Finally, applications of the microinjection technique are discussed, including gene overexpression via mRNA injection, gene silencing by delivery of antisense morpholino oligonucleotides, and the generation of transgenic zebrafish using specially engineered plasmid DNA.

Introduction to Microinjection of Early Zebrafish Embryos

One of the major advantages to working with zebrafish (Danio rerio) is that their genetics can be easily manipulated by microinjection of early stage ...

منهج في Microinjection من

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