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Mouse CRISPR/Cas9 RNP electroporation

eagle-i ID

http://eagle-i.itmat.upenn.edu/i/0000016d-d5d8-138a-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 mice was to induce a site-specific mutation in mouse embryonic stem (ES) cells, then microinject the ES cells into blastocysts (see ES cell Microinjection). This work is extremely 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 create 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 a cut has been made it is possible to:; insert, delete, or alter a specific sequence of interest. Electroporation of the Cas9 RNP is typically employed for all mutations such as small insertions, deletions or sequence changes. While some labs may prefer to prepare their own CRISPR reagents, we can also recommend ordering the guides through a commercial vendor such as IDT. The RNP will be electroporated with 0.5day gestation zygotes. The electroporation is performed using a Gene Pulser Electroporator and 1mm cuvettes. This procedure allows the RNP complex to pass through the chemically thinned zona pellucida, and penetrate the fertilized zygote where it incorporates into the mouse genome. Shortly after electroporation the 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 and screened for integration of the RNP sequence. Mice 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 facility will schedule your 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 strains tend to work better than inbred strains. A minimum of 90 electroporated zygotes are transferred into recipient mother mice, the majority of which will become pregnant. A cost saving can be offered if 2 lines are generated during the same electroporation session. Both lines can be for your own lab, or you can split the service fee with another investigator. If the core does not generate any founders from the first electroporation 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 electroporations 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
    2019-10-16T14:35:46.398-04:00
  4. creator
    ggrant (Gregory Grant)
  5. modified
    2019-10-16T14:35:50.475-04:00
Copyright © 2016 by the President and Fellows of Harvard College
The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016