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Rat CRISPR/Cas9 mRNA Microinjection

eagle-i ID

http://eagle-i.itmat.upenn.edu/i/0000016d-d5f0-8d38-eaa8-15d780000000

Resource Type

  1. Material modification service

Properties

  1. Fee for service
    Yes
  2. Resource Description
    The conventional and traditional method for creating gene-targeted rats was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts. This work is laborious and expensive. Genetic modification technology has evolved and the bacterial method of genetic repair has been adopted as a faster and cheaper method for creating gene target mutations in animals. This newly adopted system is referred to as the CRISPR/Cas9 method. This technology consists of two main components; the guide RNA (gRNA) that binds to DNA and the enzyme Cas9, which cuts the DNA at a specific location. Once DNA has been cut it is possible to insert, delete, or alter a specific sequence of interest. Microinjecting Cas9 mRNA into zygotes is the preferred method for large gene sequence modifications or conditionals mutations. While some labs may prefer to prepare their own CRISPR reagents, the Transgenic Core Lab also recommends using the Penn School of Medicine CRISPR/Cas9 Mouse Targeting Core or ordering the guides through a commercial vendor such as IDT. Your mRNA will be injected directly into the cytoplasm of a minimum of 100 fertilized 0.5d.p.c. zygotes. This can be performed in a wild-type or existing mutant strain. The mRNA is loaded into the ultra-fine injection needle and using a constant, regulated flow of pressure is then injected into each zygote. Following an incubation period, the surviving zygotes are surgically transferred into the reproductive tract of 0.5day pseudo-pregnant surrogate females. When the offspring reach 10-14 days old they will be tail snipped, the tissues will be delivered to your lab or the CRISPR Core and screened for integration of the mRNA sequence. Rats identified with the correct sequence are considered the founders, or F0; these will be transferred to your animal holding room, where they can be bred to expand the line for experimental use. The Transgenic Core will schedule strain production requests on a first-come, first-served basis. We will attempt to accommodate any strain background requests, but please be aware that hybrid/outbred strains tend to work better than inbred strains. A minimum of 50 microinjected zygotes are transferred into recipient mother rats, the majority of which will become pregnant. If the core does not generate any founders from the first microinjection session a second session will be scheduled. The fees associated with the additional animals will be billed to you, but the core will absorb the service fee. If no founders are identified from the second set of microinjections we will need to arrange a meeting to evaluate the project.
  3. Service Fee URL
    https://corelabs.research.chop.edu/sites/default/files/inline-files/Transgenic_Fee_Schedule_9.25.19.pdf
  4. Service Provided by
    Transgenic Core (CHOP)
  5. Website(s)
    https://corelabs.research.chop.edu/transgenic/services
 
RDFRDF
 
Provenance Metadata About This Resource Record
  1. workflow state
    Published
  2. contributor
    ggrant (Gregory Grant)
  3. created
    2019-10-16T15:01:46.189-04:00
  4. creator
    ggrant (Gregory Grant)
  5. modified
    2019-10-16T15:01:49.406-04:00
Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016