Production of Gene Targeted Mice

•  Introduction

Gene targeting is a technique utilizing homologous recombination between an engineered exogenous DNA fragment and the genome of the mouse embryonic stem (ES) cells. Recombination between identical regions contained within the introduced DNA fragment and the native chromosome will lead to the replacement of a portion of the chromosome with the engineered DNA. These modified ES cells can then be injected into mouse blastocysts where they can incorporate and contribute to the fetal development along with the blastomeres from the ICM (inner cell mass). These techniques can be used to ablate (knockout) gene function throughout the mouse, in selected tissues, or at specific time points of mouse development. They can also be used to introduce mutations into the genome at a desired location.
Essentially all gene targeting experiments have the following steps:

  1. construction of targeting vector containing regions of identity with the mouse chromosome (homology units or arms), a selectable marker (generally a cassette that confers neomycin (G418) resistance) and planned modifications that ablate or alter the expression of the targeted gene or region of chromosome
  2. introduction of the linearized targeting vector into mouse ES cells and selection and screening for those targeted ES clones that have integrated the planned modifications by homologous recombination
  3. microinjection of targeted ES cells into blastocysts to generate mice chimeric for the targeted ES cells and host blastocyst cells
Blastocyst Injection of Modified ES Cells
Depending on your needs, the Transgenic Animal Facility is able to perform any or all of the steps listed above: planning and construction of gene targeting vectors, targeting ES cells or blastocyst injection.

•  Getting Started

Before proceeding with any steps (Vector Design, Gene Targeting or Microinjection) the investigator will need to meet with the Transgenic Animal Facility.  This consultation will enable us to determine the specific needs of the investigator, review the targeting procedure and discuss fees for the various services that the investigator wishes the GEAM to provide. Please contact Kathy Krentz (kjkrentz@wisc.edu), (608) 890-3785 to schedule an initial meeting.

 

•  Gene Targeting Vectors

Checklist:

  1. Set up an initial meeting with us as you are beginning to design your targeting vector.   We'd be happy to make recommendations, and may be able to provide you with useful vectors or sequences.
  2. complete a billing request for services provided.
  3. Cre/loxP technology is covered by patents and license agreements. UW investigators should contact Mark Doremus, 262-0153 or mdoremus@rsp.wisc.edu to request addition to the Addendum of Authorized Users of Cre/loxP Technology.

•  Guidelines for Vector Design

We recommend incorporating the following features in your targeting vector.

  1. DNA from mouse strain 129/Sv to maximize homologous recombination frequency in 129/Sv ES cells.  If you choose to use ES cells from another mouse strain, the genomic DNA used in your targeting vector must be from the same strain (isogenic).  The ES cells we routinely use are R1 ES cells (Nagy et al., 1993).  However with sufficient time to make other arrangements, it may be possible for us to obtain and use ES cells from another strain of mice.
  2. NEO as positive selection and HSV-TK as negative selection
  3. five or more kb of genomic DNA in the two homology arms, with a minimum arm size of 1kb
  4. a unique restriction site that will be used to linearize the targeting vector at the junction between one of the homology arms and the cloning vector
  5. mutations or loxP sites should be placed as close to the positive selection cassette as possible
  6. We highly recommend using Southern blotting to identify correctly recombined ES clones. Unique probes and restriction enzyme sites that lie outside the homology regions will be used in Southern blot screens to identify correctly recombined NEOr ES clones.

•  Custom Design or Production of Targeting Vector

GEAM staff expert in molecular biology techniques can construct targeting vectors for your project for a time and material recovery fee.  We will work with you to design a vector suitable for your experiment using the guidelines described above in Guidelines for Vector Design.  We have designed and produced knock-out, knock-in, and conditional knock-out vectors for a number of UW-Madison investigators.

•  Gene Targeting

Checklist:

Prior to scheduling your targeting: You will

  1. meet with us to review your targeting vector and strategy for screening your ES clones to identify correctly targeted clones
  2. complete a billing request form
  3. obtain a MTA for R1 ES Cells
  4. initiate or obtain an Animal Use Approval or provide your Animal Welfare Issuance number
  5. provide us with an isolated bacterial colony containing your targeting vector and a detailed restriction map of the targeting vector

Linearized targeting vector will be introduced by electroporation into R1 embryonic stem cells (Nagy et al., 1993) ES cells that integrate the targeting vector either by homologous or random integration will be selected by growth on G418.  Negative selection with gancyclovir will select against clones that contained the HSV-TK cassette.  This will enrich for clones that integrated the Neo cassette by homologous recombination, and select against random integration events. Up to 480 Neor, GANCr colonies will be picked, duplicated and frozen in 96-well dishes.

DNA will be prepared from the duplicated 96-well plates and given to the investigator for genotyping.  We highly recommend using Southern blotting with probes that lie 5' and 3' to the regions of homology included in the targeting vector.  The investigator will also be responsible for verification, by DNA sequence analysis, of any loxP sites, FRT sites or specific mutations introduced by the targeting vector.

Correctly targeted clones will be amplified, frozen in cryovials, and stored in liquid nitrogen.  A sample of genomic DNA will be made from each clone and given to the investigator for re-genotyping and sequencing analysis.


Once the clones have been reconfirmed by Southern blot we recommend karyotyping be done to identify ES clones with a normal chromosome number.  We currently subcontract this service.  Karyotypically normal ES clones are ready for microinjection.

•  Microinjection of ES Cells

Checklist:

When you are ready to have your ES cell clones injected: You will need to 

  1. contact Kathy Krentz (kjkrentz@wisc.edu) to discuss the timeline for your project.  Clones are injected in the order in which they are submitted, so it is important to contact us ASAP to provide all required forms and information.  Clone must be amplified and re-genotyped prior to submission (i.e. clones must be ready to go).
  2. complete a billing request form
  3. provide an IACUC approved animal protocol number
  4. provide evidence that clones to be injected (or the lab that they were targeted in) are mycoplasma free
  5. provide the clone or clones to be injected.  Clones may be provied in frozen vials at least one week prior to injections or in culture on 35 mm plates on the week of injection. It is recommended that clones be karyotyped prior to submission for microinjection as this greatly increases your chances for success.

Targeted ES cells are grown for three to four days on a LIF (leukemia inhibitory factor) producing feeder layer.  ES cells are disaggregated into single-cell suspension, separated from the feeder cells, and injected into the blastocoel cavities of expanded blastocysts.  At this point, since most investigators choose to use ES cells derived from Strain 129/Sv mice to generate their knockouts, the ES cells are injected into C57BL/6 blastocysts which have been demonstrated to provide an optimal environment in which the 129/Sv ES cells can compete effectively with the ICM to produce high quality chimeras.   However, if investigators have access to proven ES cells from another strain, the GEAM can work with them to identify and obtain a suitable host strain assuming they are prepared to accept any additional costs or risks that may be incurred.


Following the microinjections, the blastocysts are allowed to recover and transferred into the oviducts of pseudopregnant recipients.  Pups should be born 19 days later and coat color can be identified one week after that.  Since the 129/Sv ES cells are from an agouti strain, and the blastocysts are from a black strain, the more agouti that is observed in a chimera, the better that founder is judged to be.  The founder chimeras will be ready to mate to C57BL/6 partners at 6-7 weeks of age.  Germline transmission will result in the production of agouti F1s.  Up to three chimeras from each clone will be bred in the GEAM.  Any additional chimeras will be shipped to the investigator for breeding.


•  Long-term storage/recovery

ES cell clones are stored in GEAM. A $80.00 yearly liquid nitrogen storage fee is charged per Principal Investigator in December regardless of the number of samples stored.

Investigators can request their ES cell clones at any time. ES cell clones will be processed according to Principal Investigator wishes.