How are the cells prepared?

Primogenix’ mouse embryonic stem cell (ESC) lines are expanded to passage 10-12 and sold as cryopreserved, frozen aliquots of at least 2.0x106 cells/cryogenic vial.

Primogenix’ ESC lines are carefully expanded from low passage stock vials taking care to maximize the production of high quality cultures. They are kept in log phase during the expansion. The cells are subcultured every other day at a dilution that does not exceed 1:6 throughout the expansion. Working passage lots are prepared in batches of 50-200 vials each.

The quality of the Primogenix cells is more important than the number of vials prepared from any individual batch.

Return to top

What testing is performed?

Each batch of cells is tested for:

• Cryopreservation efficiency
  • Test thaw of random vials to assess cryopreservation procedure
• Sterility
  • Growth in the absence of antibiotics
• Pathogen tested
  • IMPACT* (RADIL, University of Missouri, see below*)
  • Including mycoplasma species
• Karyotyped, by G-banding
  • 20 cells are counted and three are analyzed to determine the karyotype of the batch

Return to top

What are the culture methods?

The culture protocols are included with each shipment and can be found on Primogenix website under “Protocols”.

Note: The culture media formulation varies with the cell line. It is strongly suggested that the medium formulation provided with each line be utilized in your experiments. At a minimum, it is advised that the initial thaw and first passages be made following the protocol provided and 3-5 stock vials prepared before transferring the cells to your desired medium.

All Primogenix mESC lines have been derived by co-culture with primary mouse embryonic fibroblast (MEFs) feeder cells and in medium containing mouse LIF.

Media formulations: “Primogenix culture protocols”

Sera: HyClone ESC qualified FBS: www.hyclone.com

Serum Replacers:

AdvanceStem Serum Replacement: www.hyclone.com

Knockout Serum Replacer: www.invitrogen.com

Incubator settings:

7.5% CO₂ in humidified air, 37°C

• In classical DMEM or IMDM formulations: incubator set to 7.5% CO₂ in humidified air at 37°C.

5% CO₂ in humidified air, 37°C

• If using a lower osmolar medium such as AdvanceStem Low Osmo DMEM (HyClone) or Knockout DMEM (Invitrogen): 5% CO2 is sufficient to provide physiological pH.

Return to top

Can Primogenix mouse embryonic stem cell lines be grown in serum replacement products?

Serum replacers can be used to culture the Primogenix mESCs, however, we have not confirmed germline transmission for most of the lines following culture in medium that has only SR and does not contain FBS.

The addition of 5% SR to the growth medium (15%FBS and 5%SR) seems to have a positive effect on the appearance of the cultures and has produced germline competent chimeras.

Return to top

Do I have to use recommended media (DMEM4SC, IMDM4SC)?

One can use classical DMEM and IMDM basal media. However, there are many formulations for these “classical” medias. Compare the media formulations to the “4SC” formulations to make sure that they are identical.

Return to top

How have the cells been tested for pluripotency?

• Germline chimeras
• Immunocytochemistry: SSEA-1 and Oct3/4
• Alkaline phosphatase

Return to top

Have the Primogenix mouse embryonic stem cells gone germline?

Each of the mESC lines that are posted on the website has been proven to go germline.

We are testing our other lines and strains for germline transmission.

Microinjection of mouse embryonic stem cells (mESCs) into mouse blastocysts to determine the efficiency with which the mESC line produces chimeras and the efficiency with which those chimeras Go Germline is the “gold standard” used to assess the quality of mouse ESC lines.

Primogenix’ mouse embryonic stem cell lines are stable and robust in culture and efficiently transmit to the germline when cultured as described in the accompanying protocols.

Primogenix is developing cell lines from a variety of mouse strains. We have over 100 individual mESC lines, inquire to see if we have a line that you are interested in.

Return to top

What is up with all the mouse strains?

There are hundreds of inbred mouse strains and thousands of genetically engineered strains. Each inbred strain is genetically unique and has a unique phenotype. A major project is underway to characterize the most commonly used strains with the goal to determine how genetic variation influences complex heritable traits. The data collected is to be deposited in the Mouse Phenome Database at the Jackson Laboratory (MPD; www.jax.org/phenome).

Having said this, to date, only a few strains have been used to make knockout mouse models. This is because of the difficulty in deriving stable germline competent ESCs from strains other than 129 or B6.

Substrains of 129 and C57BL/6 have arisen over the decades; check out the Jackson website for more information on this important topic.. http://jaxmice.jax.org/geneticquality/substrains.html

Things to consider when choosing a mouse embryonic stem cell line.

One needs to choose the ESC line carefully.

Strain and substrain differences

Strain and substrain differences can have a big affect on the outcome of your experiment. Choose the strain carefully and consider the phenotype you are studying.

Growth rate and morphology

Cells should grow quickly doubling every 10-14 hours.

Colonies should be tightly clustered and when viewed with phase/contrast microscopy should have phase bright borders. The cells should have high nuclear to cytoplasmic ration.

Karyotype

The line should have a normal and stable karyotype. When reading your karyotype reports, beware of lines containing many polyploid cells and watchful for the development of clonal variants in the culture, these abnormalities complicate in vitro assays and severely decrease the efficiency of germline transmission.

Karyotype should be greater than 70% normal

Targeting vector: Isogenic DNA
If targeting a gene locus, it is advisable to consider using DNA isolated from the same mouse strain/substrain to improve targeting efficiency especially if one is using standard targeting technology and vectors in the 6-20Kb range.

Return to top