Transgenic Core Facility, Biocenter Oulu
University of Oulu
Ordering Form for Gene Targeting in ES Cells/Generation of Chimeric Mice
Send to: reetta.vuolteenaho(at)oulu.fi, jonna.ranta(at)oulu.fi or raija.soininen(at)oulu.fi
Researcher/contact person:
Research Group:
E-mail address:
Telephone number:
Telefax number:
Departmental affiliation and address:
Services are paid via:
University of Oulu internal billing
External billing
Billing address:
Purchase order number if needed:
VAT ID:
Name of the DNA construct:
Short description of the construct including the information on selection markers:
PCR genotyping design (see below):
Probe and restriction digestions for Southern blot analysis:
Linearized with:
Quantity:
Background of the targeting construct:
Preferred ES-cell line (Sv129, C57/BL6):
Animal ethical committee permit number:
Note: The researcher is responsible for the costs of care of the mice after weaning and planning the
breeding of chimeras.
Date and Signature:
The electroporation is not performed unless a gel picture of the PCR assay and a documentation (X-Ray film
/phoshoimager print) of Southern analysis have been presented. The colonies are frozen in 24-well plates, and a portion
grown for DNA analysis. PCR screening is recommended for preliminary screening, and successful targeting in PCR
positive clones is verified by Southern hybridisation. Cells of two targeted clones will be injected into C57BL/6
blastocysts to generate chimeric mice. Aggregation with CD-1 morulae can also be performed.
GENOTYPING OF ES CELLS
1) PCR
The ES cells are selected for G418 resistance: colonies that have integrated the construct into their genome survive. PCR analysis is
performed to detect clones with homologous recombination. PCR primers for the wild type allele are used for DNA quality and
quantity check. One primer outside the targeting construct, one primer in the region that is deleted in targeting. Primers for the
mutant allele: One primer outside the targeting construct (same as for wt), one primer within the neo gene or in the mutated area. Test
PCR conditions for the wt allele, using ear sample DNA as a template. For the mutant, choose a primer with similar properties.
2) Southern blot
Southern hybridization of total genomic DNA can be tricky, therefore conditions have to be well tested beforehand. Probe is chosen
outside the targeting construct and must not contain repetitive sequences. There has to be a size difference in the fragment probe
detects in the wild type and mutant allele. Prepare enough probe DNA for all the hybridisations needed. Use 20 micrograms of
genomic DNA in the restriction digestions. Reliable enzymes that usually work are BamHI, EcoRI, HindIII, KpnI, PstI, XbaI, but
others can work as well. Use fresh isotope (close to the reference date) for labelling to get a strong signal.
GENOTYPING OF MICE
Ear samples are taken from 2-week –old pups. The same PCR assay as for ES cells can be used for genotyping. Usually results are
verified by Southern for the first pups but not continuously.
The researcher is responsible for genotyping and the results should be sent to the animal facility within two weeks. Contact person at
the Laboratory Animal Centre is Eija Nissinen (eija.nissinen(at)oulu.fi). Note that the researcher pays for the animal care after
weaning!