We inject your DNA or BAC solution to the pronucleus of zygotes.
Typically we inject DNA solutions at a concentration of ~3 ng/µl, BAC solutions at ~1 ng/µl.
Your stocksolution should have a concentration of 50-100 ng/µl, solved in microinjection buffer ( 5mM Tris, 0.1 mM EDTA in ultrapure water, pH 7.4, 0.22 µm filtered). We dilute it accordingly.
For BAC DNA, additionally a polyamine mix (spermin, spermidine) and NaCl are added.
CRISPR/Cas9 mixes are injected either into the pronucleus or the cytoplasma of zygotes.
Please submit the Injection Request form "Inj request_DNA_BAC" or "Inj request_CRISPR".
Superovulation: To get enough zygotes on an injection day we treat female donor mice with gonadotropins (Inducing Superovulation).
- PMSG (pregnant mare's serum gonadotropin) is used to mimic the oocyte maturation effect of the endogenous follicle-stimulating hormone (FSH)
- hCG (human chorionic gonadotropin) is used to mimic the ovulation induction effect of luteinizing hormone (LH)
Typically we inject 5 IU of PMS on an afternoon around 4pm, and 5 IU hCG 46-48 hours later. Then females are mated to stud males.
Strain and age (=sexual maturity) of the females have a great influence on the success of superovulation.
Good results are achieved with females around 4 weeks old.