Pronuclear Microinjection |

PRONUCLEAR MICROINJECTION

Introduction

Transgenic mice are most commonly produced by microinjection of DNA into the pronuclei of fertilized single-cell mouse embryos. Transgene integration is random, with multiple copies of the transgene typically integrating into a single chromosomal locus in the embryo. If integration takes place prior to the first nuclear division, then all cells will carry the transgene. After injection, the eggs are surgically transferred to the oviducts of time-mated pseudopregnant foster mothers, generated by mating females with vasectomized males. The offspring resulting from injected eggs may or may not carry the transgene. On average, about 10-20% of the mice born will test positive for the transgene. The mice that do carry the transgene are called founders.

Service Description

purchase and housing of mice used to produce embryos for injection
superovulation of embryo donors and mating with stud males
creation of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection
harvesting of embryos from euthanized donors
dilution of the client’s transgene DNA with injection buffer, and injection of this solution into one pronucleus of each fertilized embryo
surgically transfer all embryos that survive the injection process into the oviducts of pseudopregnant foster mothers
monitoring of foster mothers before and after birth of pups
weaning, ear-tagging, and tail-tipping pups
provide tail biopsies to the client for genotyping
arrange and assist with the transfer or shipment of transgenic mice to the investigator’s mouse colony

Transgene Construct Preparation

The investigator is responsible for designing and preparing the transgene construct. For an extra fee, the TRP offers transgene design, DNA preparation, and genotyping through our partnership with the UW Mouse CRISPR Core.
The investigator should provide 30 µl or more of transgene DNA fragment at a minimum concentration of 50 ng/µl in filtered microinjection buffer (5mM Tris, pH 7.4; 0.1 mM EDTA in ultra-pure water)—see Protocols page for more information.
The construct should be designed so that the transgene insert can be purified away from plasmid backbone by gel purification.
BAC constructs can be injected in circular form or linearized.
The cleanliness of the construct is of utmost importance. It should be absolutely free of any phenol, chloroform, ethidium bromide, high salt and ethanol. Even trace amounts will quickly lyse the injected embryos or interfere with the development of the implanted embryos. "Sticky" DNA preps will clog the injection needles and kill embryos. Great care should be taken when preparing the injection mix as the success of the procedure is highly dependent upon the quality of the DNA being injected into the embryos. See Protocols page for more information.

Genotyping

The investigator is responsible for genotyping all pups.
The investigator should establish a PCR protocol that can reliably detect your transgene at the single copy level when mixed with wild-type mouse DNA. This should be completed before the construct is injected into mouse embryos.
For an extra fee, the TRP offers transgene design, DNA preparation, and genotyping through our partnership with the UW Mouse CRISPR Core.

Mouse Strain

The standard background strain used for the embryos is B6D2 (C57BL/6 x DBA/2J) or B6C3 (C57BL/6 x C3H). Embryos are produced by mating B6D2 females to C57BL/6 males, generating potential founder animals which are 75% C57BL/6 and 25% DBA/2J. Other strains can be used as egg donors, but these may require additional fees. Some strains do not respond to superovulating hormones and/or have poor quality embryos and should be avoided if possible. Pure C57BL/6 embryos can be used for injection, but due to poor survival, it is often necessary to perform multiple injection sessions in order to produce founders.

Prices and Guarantees

We guarantee that at least 70 injected embryos will be surgically implanted into pseudopregnant recipients, or 2 founder pups will be produced, whichever comes first when injecting hybrid embryos. The construct DNA provided by the investigator must be clean and non-toxic or the guarantee is void.
In the event that an alternate host embryo strain is required, the additional costs incurred from purchasing and housing a special strain of stud males, will be charged to the investigator. The investigator will also pay the difference in purchase price between the cost of standard hybrid strains, and the cost of the embryo host strain selected. No guarantee will be made as to the number of injected embryos transferred, due to strain variability in embryo production.
We will inject pure C57BL/6 embryos at no extra cost, but without guarantee. This strain tends to be less efficient due to high mortality post-injection, therefore two or three injection sessions are typically required to achieve the same results as one session using hybrid embryos.
Cancellation of injection within two weeks of the scheduled date will result in a $500 fee to cover the cost of embryo donors, hormones, and technician time.
Click here for current rates.

Reserve a Pronuclear Microinjection Session

To schedule a Pronuclear Microinjection session, go to our TRP Forms page. Fill out a “Request for Pronuclear Microinjection” form and e-mail it to Robert J Hunter. Be sure to include a current budget number and your IACUC animal use protocol number.