Transgenic Resources Program
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Gene-targeted mouse ES cells are produced by electroporating a gene targeting construct into wild-type ES cells. The targeting construct typically contains selectable markers surrounded by two regions of homology to the targeted locus. In a fraction of those cells that take up the targeting construct, homologous recombination between the genome of the ES cell and the two regions of homology will result in the replacement of the targeted locus with the targeting construct.
Knock-out or knock-in mice are created by microinjecting these gene-targeted mouse ES cells into mouse blastocysts. The injected blastocysts are then transferred to pseudopregnant females and allowed to develop to term.
The gene targeting service includes the following:
- electroporation of your targeting construct into wild-type ES cells using an Amaxa Nucleofector Device
- growth of cells under antibiotic selection
- picking up to 96 drug resistant clones
- splitting clones into two sets of 24-well plates-one set will be frozen at -80°C and the other will be provided to the investigator for genotyping
- thaw and expand clones carrying a homologous targeting event
- provide investigator with a 10 cm confluent analysis plate for Southern analysis (one plate for each clone carrying a homologous targeting event)
- freeze and store duplicate clones in LN2 to use for future experiments/microinjections
- It is the investigator’s responsibility to design and engineer the targeting construct.
TARGETING CONSTRUCT DESIGN
Gene targeting constructs are designed to undergo homologous recombination into a specific locus chosen by the investigator, usually with the aim of disrupting the gene to prevent transcription of a functional mRNA (a knock-out), or mutating the gene (a knock-in). There are many ways to design a targeting construct. Below are some general considerations:
- In order to ensure that no protein is made from the targeted gene, construct a targeting vector that will delete as much of the coding sequence as possible.
- The arms of homology should be cloned from the same strain as the targeted ES cells. We recommend targeting to C57BL/6.
- The longer the arms of homology, the better. A common strategy to aid in genotyping is to design one arm at 5-6 kb and the other arm at 1-2 kb for an overall length of 7 kb.
- Alternatively, CRISPRs can be designed to target your gene of interest with much higher efficiency compared to the classic approach to gene targeting. The CRISPR/Cas9 system works by binding specific sequences of DNA and causing a double stranded break (DSB). The DSB will be repaired by non-homologous end joining (NHEJ), which is an error-prone process leading to nucleotide deletions and/or insertions at the site of the DSB, therefore knocking-out the gene. Modifications to the gene can be made by including a repair template (a targeting vector with 500bp arms of homology), which leads to repair via homologous recombination. See Addgene’s website for further information and useful databases for designing your CRISPR.
GENE TARGETING CONSTRUCT PREPARATION
- The investigator should provide 30 µg of linearized targeting construct DNA at a concentration of 1 µg/µl.
- The DNA should be absolutely free of contamination from ethidium bromide, ethanol, phenol, chloroform, and high salt.
- Design a genotyping protocol (Southern Blot Analysis and/or PCR) that can distinguish homologous recombination events from random integration events.
ES CELL LINES AVAILABLE
The TRP has three separate ES cell lines available for gene targeting experiments.
- G4 ES cells: http://www.pnas.org/content/104/11/4455.full.pdf+htmlThis line was created by Andras Nagy from a hybrid of 129S6/SvEvTac x C57BL/6Ncr. This is our preferred line for gene targeting experiments and is available to all University of Washington investigators with a signed MTA (pdf link)
- R1 ES cells (129X1/SvJ x 129S1)
- JM8.F6 (C57BL/6N)
PRICES AND GUARANTEES
- The charge includes ES cell transfection, drug selection, picking clones, plates for storage at -80° C, duplicate plates for analysis, thaw and expansion of targeted clones for freeze at -196° C and one 10cm plate per targeted clone for Southern Analysis. Because different selection vectors included within the targeting construct result in widely variable enrichment for targeted events, no guarantees as to the number of clones arising from electroporation can be made. However, if 50% or more of the successfully targeted clones are lost through the freezing process the transfection process will be repeated at no additional cost.
- It is the investigator’s responsibility to establish a reliable genotyping protocol (PCR and/or Southern Blot) before the electroporation process is started. Clones must be screened promptly. ES cell clones that are stored at -80° C for more than 1 month will not be guaranteed to produce high-percentage chimeras.
- Click here for current rates.
RESERVE A TRANSFECTION SESSION
To order an ES cell transfection session, go to our TRP Forms page. Fill out a “Request for ES Cell Transfection” form and deliver it along with your gene targeting construct to the Health Sciences Building, room T-140, during normal business hours. Ask for Serina Tsang firstname.lastname@example.org or Bob Hunter email@example.com.