Generation and analysis of gene modified mice

Biocenter Oulu Transgenic Core Facility provides services in generation and archiving of gene modified mice.

1. Gene targeting and culture of embryonic stem (ES) cells.
2. Injection of ES cells into blastocyst stage embryos.
3. Generation of transgenic mice by DNA microinjection into zygotes.
4. Rederivation of contaminated mouse lines.
5. Cryopreservation of mouse lines.
6. Recovery of cryopreserved mouse lines.
7. Generation of induced pluripotent stem (iPS) cells.
8. Isolation of ES cells.
9. Lentivirus injections into zygotes.
10. Mouse phenotyping.
11. EMMA service: Archiving and distribution of mouse lines
     

• Service prices 2013

Contact persons:

Coordinator Raija Soininen, PhD, Docent
tel. +358-(0)294 486135; fax +358-(0)8-537 6115
e-mail: raija.soininen (at) oulu.fi

Researcher Reetta Vuolteenaho, PhD
tel. +358-(0)294 486133; fax +358-(0)8-537 6115
e-mail: reetta.vuolteenaho (at) oulu.fi

Laboratory supervisor Anniina Markkula, M.Sc.
tel. +358-(0)294 486133; fax +358-(0)8-537 6115
e-mail: anniina.markkula (at) oulu.fi

1. Gene targeting and culture of embryonic stem (ES) cells

The researcher provides the linearized construct DNA to the core facility and plans and tests the genotyping protocol. Currently ES cells in use are derived from 129S6, 129X1, and C57BL/6N strains, all with high germ line efficiency. ES cell culture, electroporation, selection, colony picking and freezing, as well as isolation of DNA from cell clones is performed by the Transgenic Core Facility staff. The researcher is responsible for the DNA analysis. Clones with the targeted alteration are expanded and frozen for further use.

Price for gene targeting: Researchers from University of Oulu: 1300 €; Researchers from other Universities: 3600 €.

The price is for one construct and covers the whole procedure from culturing and electroporation of the ES cells, picking up to 500 colonies, freezing the stocks in 24-well plates, to the isolation of the DNA of the clones. The success in the targeting is dependent on the gene region and the targeting construct, therefore, a positive result cannot be guaranteed but is in most cases obtained.

Price for the expansion of ES cell clones: Researchers from University of Oulu: 70€; Researchers from other Universities: 550€.

The price includes the thawing of 1-4 cell clones, followed by the culture and expansion into 3x3 cm plates, and freezing in vials for long-term storage in liquid nitrogen.
 

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2. Injection of ES cells into blastocyst stage embryos

The core facility prepares the ES cells for injection. If the ES cells were grown outside the core facility, a negative mycoplasma test result is required.

Superovulation of mice, collection of embryos, injections of ES cells into C57BL/6 blastocysts or morulae, and transfer into recipient females are performed by the core facility staff. If C57BL/6 ES cells are used, the core facility personnel genotypes the pups to identify the chimeric mice. With other cells, the fur color is the indicator for chimerism. Chimeric mice are delivered to the investigator or if agreed with the Transgenic core and the Laboratory Animal Center, are mated with wild type females in the barrier. The researcher pays for the animal care after weaning of the chimeras. The researcher genotypes the offspring of the chimeras and plans further breeding. Note that the researcher has to have a valid licence for generation of the mouse line before the injections can be started (ELLA).

Price for blastocyst injections: Researchers from University of Oulu: 700€; Researchers from other Universities: 2250€.

The price includes the injection of cells of one targeted ES cell clone into blastocyst stage embryos, transfer of 70-80 embryos into foster mothers and mouse care until weaning.

Price for the preparation of ES cells for injection: Researchers from University of Oulu: 60€; Researchers from other Universities: 250€.
 

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3. Generation of transgenic mice by DNA microinjection into zygotes

The researcher prepares the construct and supplies the purified DNA. For the purification protocol, click here. Superovulation of mice, collection of FVB or C57BL/6 zygotes, microinjections, and transfer into recipient mothers are performed by the core facility staff. The researcher is responsible for the analysis of the pups born, and for planning further breeding. Note that the researcher has to have a valid licence for generation of the mouse line before the injections are scheduled. The researcher pays for the animal care after weaning.

Price for pronuclear injection (FVB): Researchers from University of Oulu: 700€; Researchers from other Universities: 2500€.

Price for pronuclear injection (C57BL/6): Researchers from University of Oulu: 800€; Researchers from other Universities: 3100€.

The price includes the injection of DNA into zygotes, transfer of 200 injected embryos into transfer mothers and mouse care until weaning.
 

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4. Rederivation of contaminated mouse lines

Rederivation of mouse lines into specific pathogen free (SPF) condition is performed via embryo transfer. The researcher provides the males which are mated with superovulated wild type females. The core facility isolates, washes, and transfers the embryos into recipient females that arehoused in the SPF barrier unit. Cleansing of embryos isolated elsewhere can also be performed. If the mouse line to be rederived carries pathogenic viruses, a health screening is performed for the recipient mothers after the pups are weaned, the cost of which will be charged from the customer.

If wild type females cannot be used, the researcher has to provide 3-5 week-old females carrying the correct genotype. The core facility and the Laboratory Animal Center have to be contacted well in time for organizing the quarantine space for the mice to be cleaned.

Price for rederivation of a mouse line into SPF barrier: Researchers from University of Oulu: 250€; Researchers from other Universities: 650€.

The price includes the superovulation of 4-6 oozyte donors that are mated with the males from the mouse line to be cleaned, transfer of the embryos into foster mothers, and mouse care until weaning. Price does not include quarantine housing. The number of embryos to be transferred is dependent on the performance of the males.
 

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5. Cryopreservation of mouse lines

A mouse line can be cryopreserved by storage of sperm and/or embryos in liquid nitrogen. The researcher provides the males and sperm cryopreservation is followed by in vitro fertilization (IVF), after which two-cell embryos are cryopreserved. Sperm and embryos are stored in liquid nitrogen tanks in the core facility premises. The result of the cryopreservation is verified by a test thaw and embryo transfer.

Price for cryopreservation of sperm and the 2-cell-stage embryos generated by IVF: Researchers from University of Oulu: 650€; Researchers from other Universities: 1800€.

The price includes cryopreservation of sperm from 2-4 males; IVF with fresh and frozen sperm; freezing of 200-300 of 2-cell-stage embryos generated by IVF; five year storage of embryos and sperm in liquid nitrogen; test thaw, and transfer of the embryos into recipient females, and housing of the pups until weaning. The researcher is responsible for genotyping the offspring born from the test transfer.
 

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6. Recovery of cryopreserved mouse lines

Cryopreserved mouse lines are recovered by thawing frozen embryos or sperm. IVF is used to produce embryos if sperm is the cryopreserved material. Embryos are transferred into recipient females and the offspring is genotyped by the researcher.

Price for recovery of cryopreserved mice from embryos: Researchers from University of Oulu: 150€; Researchers from other Universities: 300€.

Price for recovery of cryopreserved mice via IVF: Researchers from University of Oulu: 650€; Researchers from other Universities: 1800€.
 

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7. Generation of induced pluripotent stem (iPS) cells

Generation of iPS cells is performed by the retrovirus infection method (Okita et al., 2007). The cells to be induced are either provided by the researcher, or the core facility will isolate and culture cells from mice provided by the customer.

Price: depends on the experiment, please contact Raija Soininen.

8. Isolation of ES cells

Isolation and expansion of ES cells can be performed from requested mouse lines using the 2i method (Nichols et al 2009). The mouse line is provided by the researcher. The core facility will isolate the morulae and isolate and culture the ES cells.

Price: depends on the experiment, please contact Raija Soininen.

9. Lentivirus injections into zygotes

Lentivirus injections are performed upon request.

Price: depends on the experiment, please contact Raija Soininen.

10. Mouse phenotyping

For more information, visit this page.

11. EMMA service: Archiving and distribution of mouse lines

The TG-core facility serves as the Finnish EMMA (The European Mouse Mutant Archive) node. For submission a mouse line to the EMMA archive, please follow instructions on the EMMA web page.

Service prices 2013

Prices for other research projects than mentioned below are given upon request. Prices do not include value added taxes and may be changed without a notice.