Transgenic and Tissue Phenotyping Core Facility

To register for an account:

To get started, you must register for an account:

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3643
2. In the upper-right-hand corner of the screen select the ‘Register using University of Oulu credentials’ option.
3. You will be directed to an authentication page where you will need to select your University and enter your HAKA credentials.
4. Once you have entered your credentials, click the ‘Login’ button.
5. You will be directed to an iLab Registration page where you will need to select your PI/Lab and verify your contact information.
6. Once your registration has been submitted, your PI will receive a notification that you have requested membership to their lab in iLab. They will need to approve your membership and assign any Cost centers for your use.

To Create an Equipment Reservation:

Once you have been accepted into your PI’s lab and assigned Cost centers, you can schedule equipment time. For equipment reservations in histology lab, you need to have the trained user status. For that, please contact TTP-core emma-oulu (at) oulu.fi.

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3643
2. At the upper right hand of the page click ‘Sign in’ and then select the ‘Sign in using HAKA credentials’ option.
3. Enter your HAKA username and password, and sign in.
4. Select the Schedule Equipment tab and choose either histology (self-use) or tissue-processor. Click on the ‘View Schedule’ button next to the instrument of interest. Click and drag on the time frame you would like to schedule your reservation for.
5. A window will pop up that will allow you to verify your reservations details before saving the reservation.

To Make a Service Request:

Once you have been accepted into your PI’s lab and assigned Cost centers, you can request services for tissue slide scanner and for services for gene-modified mice.

1. Navigate to the core page: https://oulu.ilab.agilent.com/service_center/show_external/3643
2. At the upper right hand of the page click ‘Sign in’ and then select the ‘Sign in using HAKA credentials’ option.
3. Enter your HAKA username and password, and sign in.
4. Select the Request services tab and on the Service projects & Quote Requests list click the choice of interest. Choose the initiate request and fill the request form and the payment information.
5. Verify your request service details before sending/saving the reservation.

If you are PI, make sure you do the following:

Instructions

1. Click here to log-in: https://oulu.ilab.agilent.com/service_center/show_external/3643
2. You will use your HAKA credentials to log into iLab.
3. Once logged in, look for the link in the left hand menu that says 'my labs'. Hover-over and select your lab.
4. Set the auto-approval amount if you do not wish to approve service requests below a certain euro amount. To do this, select the 'Members' panel and enter a dollar amount in the 'Auto Pre-Approval' amount and click 'save settings.’
5. To approve lab membership requests, select the ‘Membership Requests & Cost centers’ tab. New membership requests will show at the top of this page. Click “Approve” to accept a member into your lab. Click “Reject” if they are not a member of your lab.
6. To assign a Cost center to a member of your lab, find the member in the above list where it says, ‘Manage Cost centers.’ Select the checkbox(es) to the right of their name for the Cost center(s) you wish to assign them.

The BCO TTP core serves as the Finnish Infrafrontier/EMMA (The European Mouse Mutant Archive) node.

Archiving in EMMA is recommended, and it is free of charge.

For submission and ordering of EMMA mouse strains, please see www.infrafrontier.eu.

For information and prices, please contact emma-oulu (at) oulu.fi.

CRISPR/Cas9 editing: knock-out and knock-in mouse models

Microinjection/electroporation of gRNA/Cas9 protein mix and template DNA (knock-in only) into zygotes and embryo transfer are performed by the core.

Mouse backgrounds used are C57BL/6NCrl and C57BL/6J.

The customer is responsible for:

• having a valid license for generation of a new mutant mouse line (ELLA). Due to the CRISPR/Cas9 mechanisms, knock-out models are produced during the generation of knock-in models. Please, take this into the consideration in the license application.
• planning the mutation strategy, guide RNAs, and template DNA (knock-in only)
• providing the synthetic guide RNA and Cas9 protein
• genotyping of the pups born
• planning further breeding

The service includes injection/electroporation and transfer of up to 200 embryos into recipient mice, and mouse care until weaning.

Gene targeting in embryonic stem cells: knock-out and knock-in mouse models

ES cell culture and electroporation of DNA construct are performed by the core.

ES cell lines derived from 129S6, and C57BL/6N strains are available at the TTP core.

The customer is responsible for:

• having a valid license for generation of a new mutant mouse line (ELLA)
• designing and providing a linearized construct DNA. For advice on DNA preparation, please contact emma-oulu (at) oulu.fi
• genotyping of ES cells

Service includes ES cell culture, electroporation, selection, colony picking, freezing and DNA extraction from up to 200 colonies. Clones carrying the targeted alteration are expanded and frozen for further use.

Thawing and expansion of ES cell clones and freezing in vials for long-term storage in liquid nitrogen is charged separately.

The targeting success rate is dependent on the gene region and the targeting construct, therefore, a positive result cannot be guaranteed but is in most cases obtained.

ES cell injection into morula-stage embryos

ES cell culture, injection into C57BL/6NCrl or C57BL/6J morulas and embryo transfer are performed by the core.

The customer is responsible for:

• having a valid license for generation of a new mutant mouse line (ELLA)
• If the ES cells were imported to the researcher/TTP core, only cells clean from mycoplasma are accepted.
• genotyping of the pups born. For genotyping, please contact emma-oulu (at) oulu.fi
• planning further breeding

Service includes culture (mycoplasma testing of imported ES cells) and injection of one targeted ES cell clone, transfer of 70-80 embryos into recipient mice and mouse care until weaning. If C57BL/6N ES cells are used, the core facility personnel genotypes the pups to identify chimeric mice. With other cells, the fur color is the indicator for chimerism.

Chimeric mice are delivered to the customer. If agreed with the TTP core and the Laboratory Animal Center, chimeras are mated with wild type mice in the Oulu Laboratory Animal Centre (OULAC) barrier.

Transgenic models

Microinjection of a transgene construct into zygotes and embryo transfer are performed by the core.

Mouse backgrounds used are C57BL/6NCrl and C57BL/6J, for other backgrounds please, contact emma-oulu (at) oulu.fi.

The customer is responsible for:

• having a valid license for generation of a new mutant mouse line (ELLA)
• designing, preparing and purification of the DNA construct. For the DNA purification protocol, please contact emma-oulu (at) oulu.fi
• genotyping of the pups born
• planning further breeding

The service includes injection of one DNA construct, transfer of up to 200 embryos into recipient mice, and mouse care until weaning.

Cryopreserved mouse lines are recovered by thawing frozen embryos or sperm. If sperm is the cryopreserved material, IVF is used to generate embryos.

The customer is responsible for:

• having a valid license for breeding of the mouse line to be recovered (ELLA).
• genotyping of the pups born

The service includes either the IVF or thawing of embryos, transfer of the embryos into recipient mice, and mouse care until weaning.

Cryopreservation of sperm

Archiving includes cryopreservation and storage of sperm in liquid nitrogen.

The customer is responsible for

• providing 2-4 males
• genotyping of embryos or pups (in case quality control by IVF is chosen)

Service includes cryopreservation of sperm, and option for a quality control of the frozen sperm by IVF, followed by either in vitro culture or transfer of 2-cell embryos generated into recipient mice, and mouse care until weaning.

Cryopreservation of embryos

Cryopreserved embryos are generated either by IVF or by mating.

The customer is responsible for:

• providing 2-4 males (in case of IVF)
• providing both 4-6 males and 15 females (3-5 week-old) carrying the correct genotype (in case of mating)
• genotyping of embryos or pups born

In case of IVF, service includes cryopreservation of both sperm and 2-cell embryos (up to 200) generated by IVF and as a quality control either in vitro culture or transfer of 2-cell embryos into recipient mice, and mouse care until weaning.

In case of mating, service includes cryopreservation of embryos generated by mating. The project includes superovulation of 15 oocyte donors, isolation, and cryopreservation of the embryos, and as a quality control either in vitro culture or transfer of embryos into recipient mice, and mouse care until weaning.

Rederivation of mouse lines into specific pathogen free (SPF) condition is performed via embryo transfer. Cleansing of embryos isolated elsewhere can also be performed.

In case the mouse line is imported, please contact OULAC https://www.oulu.fi/keks/ for the import and housing.

Customer is responsible for:

• providing 2-4 males carrying the correct genotype. If wild type females cannot be used the customer provides 4-6 females (3-5 week-old) carrying the correct genotype.
• genotyping of the pups born

Service includes superovulation of 4-6 oocyte donors, transfer of the embryos into recipient mice, and mouse care until weaning.

For these services, please contact emma-oulu (at) oulu.fi.

• iPS cells
• Isolation of ES cells
• Ovary transfer
• Importing of mouse lines
• Shipping of mouse lines

Biocenter histology lab, located at the Kieppi building, Aapistie 5A, 4th floor, offers services, equipment and space for tissue processing, sectioning, and staining. For further information on the equipment, please see iLab; Schedule Equipment, or please, contact bcotg (at) lists.oulu.fi. Usage of the histology devices/services is free of charge.

• Tissue processor is operated by the TTP core personnel.
• Other instruments are open-access after user training. These include tissue embedding console, para trimmer, two cryotomes, two microtomes, slide printer, cassette printer, staining hood, stereo microscope with camera, two heating ovens and a vibratome.

On request, tissue sectioning and staining services are provided and charged accordingly. For further information please, contact emma-oulu (at) oulu.fi.

Slide scanner is located at the Kieppi building, Aapistie 5A, 4th floor, lab 478B. You can bring your slides on a cabinet shelf in the corridor outside the lab.

• Hamamatsu NanoZoomer S60 is for scanning mounted histological specimens using either 20x or 40x modes.
• Accepted slide size is 26 mm x 76 mm or for double sized slides 52 x 76 mm, thickness max 1,2 mm.
• Image data will not be stored after scanning but instead delivered by email using FileSender. Alternatively, data can be stored on an external hard drive/memory stick provided by the customer. Please note, that the image size can be up to 1.5 GB.

For further information, see iLab for service request or please, contact bcotg (at) lists.oulu.fi.

Visiopharm software is for image analysis-based quantification of tissue properties in more than 50 different image formats. Two licences are available: one associated with the Visiopharm PC at the Histology Lab 479B and the second one is a floating licence for remote use. For further information, please contact emma-oulu (at) oulu.fi.

• The software utilizes Analysis Protocol Packages (APPs) that are small programs to automatically analyse brightfield and fluorescent images.
• APPs are either self-made or they can be requested from the Visiopharm APP center. 50 optional APPs are freely available at the core.
• TTP core personnel provides user training and help for troubleshooting.

TTP core provides service for APP design to answer specific research questions of customers. Tailored APPs are constructed on demand.

• To get started, please contact emma-oulu (at) oulu.fi for the kick-off meeting to plan the image analysis project.
• The service fee is based on the amount of work required for designing the APP(s).
• In case of publication, the contribution of the TTP personnel shall be acknowledged.
• The TTP core retains right to reclaim APP design in further APPs.