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Mouse Embryonic Stem Cell Microinjection

eagle-i ID

http://eagle-i.itmat.upenn.edu/i/0000016d-d5dd-92f1-eaa8-15d780000000

Resource Type

  1. Material modification service

Properties

  1. Fee for service
    Yes
  2. Resource Description
    Knockout mouse models are widely used to study human diseases caused by the loss of gene function. Examples of these diseases include cystic fibrosis, beta-thalassemia and various forms of cancer. Information from studies with knockout mice can lead to a better understanding of the pathogenic mechanisms of human genetic and infectious disease, as well as provide animal models that can be used to test new genetic and drug therapies to treat these disorders. This artificially induced mutation is carried in every cell of a knockout mouse throughout development. The resulting phenotype (appearance, biochemical characteristics, behavior, etc.) may provide some indication of a gene's normal role in the mouse. Knockout mice are produced by a technique called gene targeting. This technique works by isolating and activating a gene sequence and then replacing it with a version of the same gene sequence that contains a mutation. The replacement occurs by homologous recombination, where two very similar DNA sequences line up next to each other and exchange parts. Gene targeting is carried out in mouse embryonic stem (ES) cells. Embryonic stem cells are derived from very early (usually male) mouse embryos and have the capacity to contribute to the complete development of the animal. The aim is to get modified ES cells to contribute to a germ line. Some sperm are produced that carry the desired mutation and; these will then fertilize a wild-type egg. The resulting progeny develop with one copy of the mutated gene in every cell. Interbreeding these offspring will generate some homozygous individuals that carry both copies of the mutated gene – these are the knockout mice. Prior to initiating chimera production, investigators must ensure they have provided documented evidence that the parental cell line has been screened for absence of microorganism contamination. It is important to remember that feeder cells (primary mouse embryonic fibroblasts, MEF) must also be screened for the presence of pathogens; these also have the potential to transmit pathogens to the ES cells. Additionally, the clones, containing the correct target sequence will need to be karyotyped to ensure they contain the correct number of chromosomes, to ensure they are not euploid. (A) Normal Session: Chimeric mice will be produced by injecting Embryonic Stem (ES) Cell clones, containing a specific gene targeted mutation, into the blastocyst cavity of E3.5day embryos. The injected blastocysts are cultured 1-3 hours and then surgically transferred into the uterus of a 2.5d.p.c. pseudo-pregnant recipient mother mouse. Approximately 10 days after birth the pups are identified for level of cell contribution, which is apparent by the coat color differences. The fur of chimeric heterozygous pups will be a mix of the host blastocyst strain and the ES cell strain. Mice with the highest amount of cell-derived fur will be selected for future breeding to establish the colony. These mice will be transferred to you animal holding room, where they can be bred to check for germline transmission and to expand the line for experimental use. (B) No ES Cells: Occasions may arise when the ES cells being, prepared for microinjection, are not viable, either due to contamination or growth-related problems. In this instance a lower fee has been developed, which will cover the cost of the work performed up-to the day of the microinjections. If the project needs to be repeated you will incur the full-service fee. (C) Debris with ES cells: Often when the Transgenic Core receives ES cells, for microinjection, they contain lots of debris, either from dead cells or feeder cells. When this occurs, the debris obstructs access to the viable ES cells, sticks to the outside of the injection needle and will also clog the injection needle. These problems lead to a much lengthier microinjection day and also the use of more, than typical, injection needles. This additional timing and extra supplies are reflected in the increased fee structure for this service. The above problems do not inhibit the project from being completed, they just slow the process down. The facility will schedule your strain production requests on a first-come, first-served basis. We will attempt to accommodate any appropriate strain background requests. The core will microinject approximately 40-50 blastocysts for each ES cell line. Approximately 8-15 ES cells are microinjected into each blastocoel cavity. If available the transgenic core will microinject up to 2 clones of the same targeted line per session. We expect to generate 10-20 pups from an ES cell microinjection session, this number is typically sufficient for the production of chimeric mice. Although it is common that founder mice can be generated from fewer animals too. Germ line transmission of chimeric animals cannot be guaranteed. 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, however, no founders are identified from the second set of microinjections we will need to arrange a meeting with the principle investigator 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
    2019-10-16T14:57:52.486-04:00
  4. creator
    ggrant (Gregory Grant)
  5. modified
    2019-10-16T14:58:33.592-04:00
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The eagle-i Consortium is supported by NIH Grant #5U24RR029825-02 / Copyright 2016