University of Southern California
USC/Norris Comprehensive Cancer Center
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Transgenic/Knockout Mouse Core Facility



Welcome to the USC/Norris Comprehensive Cancer Center Transgenic Core Facility.  The Facility was established in 1993 to provide high-quality embryo and stem cell manipulation, microinjection and related services to the USC community, as well as outside researchers. The core handles the production of transgenic mice, gene targeting and the production of knockout Mice, Construction targeting vector design, preparation of DNA for injection and In vitro fertilization, Re-derivation, as well as Cryopreservation.

We have more than 15 years of experience in generating transgenic and knockout mice. Thus we anticipate no problems in meeting your request. We will be happy to work with you to optimize the procedures for efficient production for ES cell gene-targeting, production of transgenic and knockout/in animals as you need in your grant proposals. We are looking forward to working with you.


Services
Order Forms Available for the following available online on website

1) Production of Transgenic Mice:  For transgenic models, we perform pronuclear microinjection with plasmid DNA fragment or BAC DNA. The Transgenic/Knockout Mouse Core Facility (TMCF) will harvest 150-200 eggs and inject DNA (prepared and purified by the investigator*, protocols are available as needed) into the pronucleus of fertilized ova. Eggs that survive to the 2-cell stage will be implanted into pseudo-pregnant females. Transgenic status refers only to DNA integration as assessed by Southern blot. We can't guarantee expression of an RNA or protein product from the integrated DNA. For embryo donors, the available strain is B6D2F1. Investigators have to pay for the donors if the special background required.

2) Knock-Out Order-ES Cell Injection: TMCF will inject 2-3 independent targeted ES cell clones (provided by the investigator) into C57BL/6J blastocysts. In total, 120 blastocysts will be injected and 12 embryo transfers will be performed. We do not guarantee germline transmission. However, our experience indicates that if the ES cells are of good quality, then the above procedure has a strong probability of producing at least two germline chimeras.

3) Gene Targeting- ES Cell Gene Targeting: Advice on vector design. Electroporation (TMCF provides ES cells). Selection of 300-400 ES cell clones. ES cell clone lysis in 96 well plates will be provided customer for analysis to identify recombinant clones. All targeted clones will be expended and provided for confirmation. Injection of ES cells into blastocysts, please see our "Knock-Out" order form 

4) Additional Services for ES Cell:

  • 1. ES Cell Cassette Deletion: This service allows for the deletion of drug selection cassettes utilizing cre or frt recombinase in vitro. 100-200 clones are picked and ES cell clone lysis will be provided for analysis.
  • 2. Southern Blotting Screening: This service allows for isolating DNA of ES cell, testing southern blotting probes, and screening ES cells by southern blot. The positive clones will be confirmed again. 3. Additional Services for ES cells Prices are not fixed, and are dependent on the services required on a case-by-case basis.

5) In Vitro Fertilization: Reconstitute live mice from fresh or frozen sperm; provides a variety of options for recovering mouse strains endangered by poor breeding, health issues or other problems. Generate a large cohort of same-aged mice in one step. Brief Procedure: The P.I. provides one or more males (females are included if special strain required) and the Core Facility will harvest oocytes from up to 8 superovulated females and fertilize them in vitro with sperm harvested from the male(s). All 2-cell stage cultured embryos will be implanted into pseudopregnant foster mothers. There are no guarantees regarding the number of pups produced, but if no pups are obtained, the Core Facility will repeat the procedure once at no extra charge.

6) Embryo Rederivation is used to:
  • 1.  Create a pathogen free strain of mice from lines that are contaminated with various pathogens (MHV, Pinworm, Parvovirus).
  • 2.  Thaw frozen embryos and bring them back to life. Brief Procedure: For mice infected with pathogens, the P.I. coordinates the receipt of at least 4 males under the P.I.'s animal care program number.  The Core acquires Wt B6D2F1 females (P.I. must provide the females if a specific background is required) and schedules the procedure for cleaning up the line via embryo transfer.  For frozen embryos, the P.I. transfers embryos in liquid nitrogen and the Core thaws the embryos, and performs implantation.

7) Embryo Cryopreservation: The storage of mouse embryos in liquid nitrogen offers a safe way to preserve lines with potential future use but no current use.  This also saves facility space and the expense associated with keeping a breeding colony.  Another advantage is that during the process of embryo collection, most pathogenic organisms are excluded.  For cryopreservation of a mouse line, the P.I. coordinates the receipt of at least 10 males ages 2.5 - 6 months of the desired strain under the P.I.'s animal care program number.  The Core acquires Wt B6D2F1 females (P.I. must provide the females if a specific background is required) and schedules the freezing 200 or more fertilized eggs from each line in straws.  As a quality control, the Core will thaw a straw of embryos along with each frozen line and will culture them to the blastocyst stage.

8) Special Services Offered (mouse):
Super-ovulation, Timed pregnancy, Blood collection, Organ collection, Embryo dissection, ovary transplantation and Fertilized embryos.



Turn Around Time

1) TRANSGENIC MOUSE Orders: We will pay for the maintenance of foster mothers and pups up to three weeks from date of birth. During that time we expect the PI to (1) Genotype the mice; (2) Transfer the mice to the PI's vivarium protocol. We will charge double for the mice left on the Core protocol after three weeks. In general, the injection will be repeated until at least 2 transgenic pups are obtained (except the cases in which the transgenic causes embryonic lethality).

2) Knock-Out Order: Blastocyst injection will be scheduled soon after we receive the order form & ES cell clones. The third clone will be injected if there is no chimera obtained from injection of the first two clones. All chimeras will be transferred to PI’s vivarium protocol in three weeks.

3) Gene Targeting: Gene targeting will be scheduled soon after we receive the order form and targeting construct. If the construct selection cassettes work well, the whole process will take four to five weeks to give ES cell lysis to PI for screening.

4) In Vitro Fertilization: In vitro fertilization will be scheduled soon after we receive the male mice. All 2-cell stage cultured embryos will be implanted into pseudopregnant foster mothers and the pups are all transferred to PI’s mouse protocol # in three weeks.

5) ES CELL: Depend on service requirement.

6) Embryo Rederivation: Embryo rederivation will be scheduled soon after we receive the males. All the newborns will be in quarantine for about 6 weeks under PI’s mouse protocol# and all confirmed Pathogen free pups will be transferred to PI’s clean mouse room.

7) Embryo Cryopreservation: Embryo cryopreservation will be scheduled soon after we receive order form. As a quality control, the Core will thaw a straw of embryos along with each frozen line and either culture them into blastocyst stage or implant into pseudopregnant females (the latter will take additional 6 -8 weeks for pups & genotype). In general, frozen embryos will be transferred to PI’s Liquid Nitrogen tank in 4 – 6 weeks.