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|  Oregon Health and Science University |
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| Oregon Health and Science University |
Currently, the transgenic facility is generating transgenic and knock-out mice, cryopreserving mouse embryos as well as performing various other services. We are available for consultations and providing letters of support to investigators.
Member:
Huang , Elaine T.
Role:
Research Associate
Incuabators are "gassed KSOM or M16 in a water jacketed, 5% CO2 incubator at 37°C and 95% humidity."
"The FTS Bio-Cool is the most reliable, safe, and easy to use controlled rate freezer for cryopreservation. Typical applications include cryopreservation of human and animal embryos and biological tissue and cell lines. The Bio-Cool is the only controlled rate freezer that does not require expendable liquid nitrogen and the associated pumping, re-filling, and storing of a high-pressure liquid. The Bio-Cool, simply plugs into a standard electrical outlet and quietly provides low temperature cooling to either -40ºC or -80ºC."
Used to produce microforge glass pipettes.
Used for blastocyst injection.
Used to produce microforge glass pipettes.
Use for embryo storage.
This incubator is used for culturing embryonic stem cells.
Consists of Zeiss Axiovert 135 inverted, epifluorescent microscope, pair of micromanipulators, CCD camera with monitor, Bionomic temperature controller for stage, Sutter pressure injector, and anti-vibration table.
"We use the UltraClean GelSpin Mo Bio kit (catalog # 12400-50) to purify all DNA constructs prior to use in our projects."
"ES cells must be kept in R1 media with LIF. Media must be changed every day and cells need to be passaged well before reaching confluence. Failure to do so results in differentiation of stem cells and renders the culture unusable."
"Embryos are harvested from naturally mated or superovulated and mated females between e12.5 and e15.5 with the optimal time being e13.5. Heads and internal organs of the embryos are removed and the remaining tissue is rinsed in sterile PBS and minced. After the addition of trypsin and DNAseI the tissue is incubated for 15 minutes and then passed through a 16G needle. The resulting mixture is centrifuged at roughly 1000RPM for 5 minutes and the supernatant is collected and spun again and the very loose pellet of cells is plated in MEF media. The supernatant is allowed to incubate for another 10-15 minutes, spun down and the resulting loose pellet is plated."
"3 to 5 week old mice are superovulated and checked for the presence of a copulation plug. After cervical dislocation, fertilized embryos are collected by (e0.5) tearing the ampulla and releasing them into a hyaluronidase/M2 solution for dissociation, (e1.5-e2.5) flushing out via the infundibulum of the oviduct or (e3.5) flushing out of the uterus. Embryos are maintained in gassed KSOM or M16 in a water jacketed, 5% CO2 incubator at 37°C and 95% humidity."
"Fur over the left lumbar area, approximately 2 cm2, is sprayed with 70% EtOH, and a 1 cm incision is made. After opening the abdominal cavity, the fat pad overlying the ovary is grasped with sterile forceps, and the ovary and distal end of the uterine horn is exteriorized. Embryos can either be transferred into the oviduct (zygotes, 2-cell embryos) or the uterus (blastocysts). For 2-cell embryos, the tip of the pipette is inserted into the infundibulum and with the application of positive air pressure, 10 to 20 embryos are placed into the fallopian tube. Transfer of blastocysts is achieved by generating a port at the distal end of the uterus using a hypodermic needle followed by transfer of 8 to 10 blastocysts into one horn of the uterus. Following embryo transfers, the abdominal wall is sutured with 3-0 or 4-0 PDS-II taper and the skin incision is closed using surgical staples or PDS-II. Post-surgical care includes a heating pad or lamp to avoid hypothermia until the mice are recovered from anesthesia."
"The abdomen is cleaned with 70% ethanol, and a 1.0 cm transverse incision is made in the ventro-distal abdomen. The fat pads overlying the testis and vas deferens are grasped and exteriorized using sterile forceps. Both vas deferentia are then cauterized, and the testes are replaced into the abdominal cavity. The incision is closed as described above. Post-surgical care includes a heating pad or lamp to avoid hypothermia until the mice are recovered from anesthesia. Animals are naturally mated or mated to superovulated females and a minimum of 2 plugged, non-pregnant females indicates a successful vasectomy."
Production of chimeric mice by injection of ES cells into blastocysts. Includes trangene microinjection into C57BL/6 and B6D2F1 hybrid oocytes.
For example, Cre-LoxP driver and reporter strains.
"We are available for consultations and providing letters of support to investigators."
"The protection of mouse models is accomplished by cryopreservation of germplasm such as embryos and sperm. Cryobanking with the core ensures that samples are stored at two geographically separate locations."
Gene targeting by homologous recombination and clonal selection or electroporation with knockout vectors into mouse embryonic stem (ES) cells.
"The facility is fully equipped with tools for standard molecular genetics service, including PCR machines, centrifuges, and apparatuses for gel electrophoreses etc. Moreover, TMM is performing standard molecular genetics service such as genomic and plasmid DNA purification. Additionally, we can amplify the fragments of your genes of interest, isolate PCR products and sequence them in collaboration with the DNA Service Core. We also have an inventory of PCR primers for the standard transgenic and reporter genes such as neomycin phosphotransferase, thymidine kinase beta-galactosidase, green fluorescent protein and others. Moreover, TMM can design PCR primers for your newly produced animals. To facilitate confirmation of genetic transmission of transgenes we can tag, collect tail samples, extract DNA and genotype your mice by PCR. Additional services are possible in collaboration with the TMM."
For transgenic mouse strains, including specific pathogen-free (SPF) mouse strains.
