Transgenic Production
Transgenic Production Services
In the planning stages of your transgenic project, please contact Pete Mathers to discuss how we can best work with you to assure the success of your project.
WVU OLAR Protocol Requirements
You must have an approved animal protocol on file with the Office of Laboratory Animal Resources. The protocol must be able to accommodate up to 135 mice for each transgenic construct. Mice used for transgenic production are ordered under the investigator’s protocol number, but are purchased by the TACF and housed under the TACF protocol. To help reduce the number of animals used, where ever possible, mice will be used for multiple transgenic projects.
Initiate a Transgenic Project
To initiate a project, you must submit a Transgenic Production Work Request form, complete with a photo of an EtBr-stained gel with markers of known concentration (i.e. Invitrogen Mass Ladder). Projects are scheduled on a first-come, first-served basis when the request form and the purified DNA for microinjection have been received. Upon receipt of the DNA you will be billed and a production schedule will be provided with a timeline for microinjection. The purified DNA for microinjection should be delivered to the TACF, room 229C. Please contact Ingrid Weterrings to schedule a delivery date and time of the DNA to the TACF.
Guarantee of Transgenic Founders
The Transgenic Animal Core Facility will make every effort to produce transgenic mice with your construct, and we guarantee at least two transgenic founders capable of transmitting the transgene to their progeny. If two founders are not produced after two days of successful injections, an additional one to two days will be scheduled as soon as possible at no additional charge. If after four days of injections no founders are produced, we will request a meeting to discuss how to proceed. The guarantee does not apply unless the DNA has been prepared by an approved protocol. We do not guarantee expression of the transgene. A founder is defined as any mouse carrying the DNA construct in its somatic cells.
Selection of Mouse Strain
The standard inbred mouse strain used the TACF for microinjection is the FVB/N. If you require microinjection on another background strain, this can be arranged for an additional charge.
Preparation of DNA for Microinjection
The DNA purity and concentration of the injected DNA are critical to the success of the project. An approved protocol for purification of construct DNA for microinjection is available. It is the investigator’s responsibility to provide high-quality DNA. Please submit 50ug isolated DNA fragment from Endofree-Qiagen preparation. Fragment must be free of plasmid backbone.
Identification of Transgenic Founders
Four weeks after the micro-injections are completed the TACF will provide the investigator with the number of pups born and the anticipated weaning date. At weaning, tail biopsies will be taken and the mice will be transferred to the investigator’s protocol. The investigator will be notified when the tail biopsies are in the freezer in the necropsy room in the HSC animal quarters. A Transgenic Rodent Screening Form will be included with the tail biopsies. The TACF should be notified of the genotyping results by completing and submitting the Transgenic Rodent Screening Form to TACF. Upon receipt of the genotyping results, if requested, the TACF will euthanize all non-transgenic animals.
Time Frame for Production of Transgenic Mice
Many factors can influence the time frame for the production of transgenic animals. This time frame represents the time needed for the production of potential transgenic founders using the FVB strain for microinjection. Upon receipt of the necessary paperwork and the purified DNA construct, a production schedule will be developed and provided to the investigator. As production proceeds, the investigator will be advised of any changes to the schedule. The anticipated production timeline from receipt of the DNA to delivery of tail biopsies for genotyping to identify potential founders is 10 to 13 weeks. The time needed for each of the stages of production is detailed below:
2 weeks
Upon receipt of the completed transgenic production request form and the purified DNA construct, animals will be ordered. Once they are received, time is required to acclimate embryo donor females. Confirmation of DNA concentration prior to microinjection will occur at this time.
1 - 2 weeks
Two microinjection days will be scheduled for each construct. As needed, additional days will be added to the schedule. Please be advised that injections are scheduled on a first-come, first-served basis, and the time frame for production of your construct may be extended. Your construct will be put into the injection schedule as soon as possible.
3 - 4 weeks
Litters will be born three weeks after the date of embryo transfer. If your construct was injected over a two-week period, you will be notified when the last litter has been born.
3 - 4 weeks
Litters are weaned at 3 to 4 weeks of age. At that time, the pups are ear tagged and tail biopsies are taken and provided to the investigator for genotyping and the identification of transgenic founders. At this time, the weaned mice are transferred to the investigator’s protocol.
1 week
This interval of time is allocated for PCR genotyping of tail biopsies. The investigator must provide the TACF with the ear tag number(s) of all founders.
1 week
Upon receipt of genotyping results, if requested, the TACF will euthanize all non-transgenic animals.