Microinjection of zebrafish embryos to analyze gene function.

نویسندگان

  • Jonathan N Rosen
  • Michael F Sweeney
  • John D Mably
چکیده

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.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Glass-capillary-accessible Dynamic Microarray for Microinjection of Zebrafish Embryos

This paper describes a glass-capillary-accessible dynamic microarray for an efficient microinjection of zebrafish embryos. The microfluidic channel has an array of holes at the trapping sites and enabled a rapid alignment of eggs, direct injection, observation, and selective retrieval of embryos. Fertilized zebrafish eggs can be loaded without pumping, and genes of interest or antisence oligos ...

متن کامل

Zebrafish Functional Genomics Development at UW-Stout

Since the completion of the human genome sequencing project, morpholino phosphorodiamidate oligonucleotide (MO) knockdown in zebrafish has been increasingly used to elucidate human gene function. As part of the effort to expand the functional genomics screening capacity at the University of Wisconsin-Stout, this project investigated MO microinjection techniques, embryo production, and brine shr...

متن کامل

Microinjection free delivery of miRNA inhibitor into zygotes

The development of gene delivery systems into embryos is challenging due to technical difficulties, delivery efficiency and toxicity. Here, we developed an organic compound (VisuFect)-mediated gene delivery system for zygotes. The VisuFect, which is hydrophilic and Cy5.5-labeled, was conjugated with poly(A) oligo (VFA). The VFA into CHO cells showed clathrin-mediated internalization and no toxi...

متن کامل

Ectopic expression and knockdown of a zebrafish sox21 reveal its role as a transcriptional repressor in early development

Sox proteins are DNA-binding proteins belonging to the HMG box superfamily and they play key roles in animal embryonic development. Zebrafish Sox21a is part of group B Sox proteins and its chicken and mouse orthologs have been described as transcriptional repressor and activator, respectively, in two different target gene contexts. Zebrafish sox21a is present as a maternal transcript in the ooc...

متن کامل

Inhibition of Xbra transcription activation causes defects in mesodermal patterning and reveals autoregulation of Xbra in dorsal mesoderm.

The Brachyury (T) gene is required for formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In this paper, we first show that the Xenopus homologue of Brachyury, Xbra, and the zebrafish homologue, no tail (ntl), both function as transcription activators. The activation domains of both proteins map to their carboxy terminal regions, and we note that the ...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:
  • Journal of visualized experiments : JoVE

دوره 25  شماره 

صفحات  -

تاریخ انتشار 2009