Skip to content
printicon
Main menu hidden.

Umeå Transgene Core Facility

Umeå Transgenic Core Facility (UTCF) is housed within UCCB and is a fully integrated platform for generation, maintenance and DNA analysis of genetically modified animals. The transgenic facility performs pronuclear injections, blastocyst injections, embryo transfer, in vitro fertilization, and sperm cryopreservation.

Overview of the different technologies

Genotyping

Client responsibilities

  • provide tissue samples or prepared DNA

  • provide primer sequences and protocols for each PCR

  • provide ordering form


UTCF responsibilities

  • prepare DNA (if client has not)

  • run PCR and visualize the result with agarose gel electrophoresis

  • send result by email, complete with gel picture within 5 working days after we receive clients samples and ordering form

Blastocyst injection

The production of knock-out mice involves the microinjection of targeted embryonic stem (ES) cells into the 3.5 day old embryo, the blastocyst. The introduced ES cells will integrate within the embryo, resulting in chimeric mice where some cells in each tissue are derived from the ES cells. If the germ cells consist of the targeted ES cells, the mutation can be passed on to the progeny.

As customer at UTCF you must provide the targeted ES cells, with a health report showing that they do not carry any mouse pathogens. UTCF will then thaw a vial of each clone to be injected, expand the cells and pass them once. After that we will perform the blastocyst injection, which includes the following steps:

  1. Collection of blastocysts

    In order to be able to visualize chimeric mice by coat color, it is advisable to use a blastocyst donor strain different in coat color to that of the ES cell strain.

    Similar to the procedure for pronuclear injections, 15 female mice start to superovulate after two injections of gonadotropins and then mated with stud males. The blastocysts are harvested by flushing the uterine horns from plugged females at 3.5 days post coitum.

  2. Microinjection of ES cells into blastocysts

    The harvested blastocysts will be injected with 12-15 ES cells into the blastocoel cavity. As the embryo develops, the introduced ES cells will integrate within the recipient embryos inner cell mass to form a new chimeric embryo.

  3. Transfer of injected embryos to pseudopregnant females

    In order for the injected embryos to develop to term, they are transferred into the uteri of pseudopregnant female recipients. These are females mated with appropriate timing to vasectomised (sterile) male mice. Once born, it is possible to determine the chimeric mice by coat color which will show as patches of different color in the fur. However, this is not evidence of ES cell integration within the germ cells of the mouse. Germ line transmission can only be determined by breeding the chimeras, and analysing the progeny.

Client responsibility:

  • Consult with UTCF coordinator

  • Provide 1 vial of prepared ES cells, 1 or 2 clones.

We also need the following information for each vial of cells:

  1. Genetic background of the ES cells (what mouse strain were they derived from)

  2. Approximate number of cells per vial.

  3. Type of medium in which the cells were cultured before freezing.

  4. Health report proving the ES cells free from pathogens

  5. Provide ordering form and approved ethical permission together with ES cells

  6. Screen resulting pups for chimeras (if not seen by coat color) or this can be done by the Genotyping facility.

UTCF responsibilities:

  • thaw and prepare the ES cells for injection

  • prepare blastocysts

  • inject ES cells into blastocysts

  • transfer injected blastocysts into a minimum of 6 foster females, or 3/clone if 2 clones were injected.

  • house born pups until weaning (around 4 weeks)

Mouse strains used:

Our blastocyst donors are C57Bl6 females mated with C57Bl6 males. If the client requests a different donor strain, UTCF will purchase this and any additional costs will be passed on to the client.

Embryo transfer

Client responsibilities:

  • consult with UTCF coordinator

  • for re-derivation of a mouse strain, provide at least 5 mating pairs for egg donation

UTCF responsibilities:

  • Dissect oviducts in order to obtain fertilized eggs

  • implant embryos into foster females, as many as possible from the number of eggs provided

  • Provide at least one born litter from the foster females

For order of Embryo transfer always consult with the UTCF coordinator.

In Vitro Fertilization (IVF)

Client responsibilities:

  • Consult with UTCF coordinator

  • Provide frozen sperm, or male(s) from which sperm will be collected

  • Provide females for superovulation

UTCF responsibilities:

  • Collect eggs from 10-20 B6/CBA females, or other background

  • Perform IVF using the provided sperm

  • Perform embryo transfer with the fertilized embryos, up to 6 embryo transfers/strain

  • House born pups until weaning

Pronuclear injection

Pronuclear injection is the most common method used to create a transgenic mouse. A transgenic mouse defines a biological model which has been genetically modified by the introduction of a foreign DNA construct into a fertilized mouse egg. The insertion of the foreign DNA usually results in expression of a new gene (gain-of-function) or in the overexpression of endogenous genes.

As customer at UTCF you must provide the linearised and purified DNA construct. After that we will perform the pronuclear injection, which includes the following steps:

  1. Collection of fertilized embryos

    In order to obtain a satisfying number of fertilized eggs, 15 female mice are stimulated to superovulated using two injections of gonadotropins and then mated with stud males. The fertilized embryos are harvested at the single cell stage after dissection of the oviduct from plugged mice at 0.5 days post coitum. There are biological variables that can influence the quantity and quality of eggs for microinjection. In particular, the mouse strain plays a key role. Hybrid strains are commonly used since they have better reproductive characteristics and enhanced egg quality compared with inbred strains. Here at UTCF we primarily use the F1 hybrid strain CBABL6/CBA.

  2. Microinjection of DNA into fertilized embryos

    For a brief window of time, the pronuclei that contain the genetic material from the sperm head and the egg is visible within the protoplasm of the single cell embryo. At this stage, the linearized DNA construct is injected into one of these pronuclei which in some of the embryos will lead to a random integration of the injected DNA in the genome. The injection of the DNA is done using a micro injector connected to a microscopes with Differential Interference Contrast (DIC) optics, which enables a clear visualisation of the pronuclei.

  3. Transfer of injected embryos to pseudopregnant females

In order for the injected embryos to develop to term, they are transferred into the oviduct of pseudopregnant female recipients. These are females mated with appropriate timing to vasectomised (sterile) male mice. They will give birth around 20 days after implantation, generally 10-20% of the born pups will have integrated the DNA construct in their genome.

Client responsibilities:

  • consult with the UTCF coordinator

  • create transgene plasmid, cut transgene from plasmid and purify the DNA

  • provide purified DNA with known concentration and a gel photo to UTCF, together with ordering form. In addition, attach your valid ethical permission.

  • screen resulting pups for positive transgenes - or this can be done by the genotyping facility.

UTCF responsibilities:

  • dilute construct to 2 μg/ml in injection buffer

  • prepare pronuclear-stage embryos

  • inject transgene into embryos

  • implant embryos into a minimum of 6 foster females

  • house born pups until weaning (around 4 weeks)

Mouse strains used:

Our egg donors are B6/CBA F1 females mated with CBA males. If the client requests a different donor strain, UTCF will purchase this and any additional costs will be passed on to the client.

Sperm cryopreservation

Client responsibilities:

  • Consult with the UTCF coordinator

  • Provide 2 males, age 12 weeks- 8 months

  • Pay a non-refundable start-up fee of 1000 SEK (20% of total cost)

UTCF responsibilities:

  • Freeze sperm from the males, 10 straws/male

  • Perform an IVF as a quality control of the frozen sperm, 1 straw/male will be used

  • Perform embryo transfer after IVF to ensure that live pups can be produced

  • Store the frozen sperm in liquid nitrogen

Price list

Blastocyst injection:                        32 500 SEK
Embryo transfer:                                6 500 SEK
In vitro fertilization:                          6 500 SEK
Pronuclear injection:                        21 500 SEK
Cryopreservation of sperm:             5 000 SEK; annual freezer cost 500 SEK
Genotyping: cost per hour:                     440 SEK + reagents and consumables. Estimated time for 30 PCR samples =3,5-4 hours

Note, the prices apply to researchers affiliated to Umeå University.

Order forms

Blastocyst injection

Genotyping

Pronuclear injection

Questions?

Contact UTCF coordinator