Genetic Engineering Facility
The NEI Genetic Engineering Core (GEC) provides NEI researchers with services to generate, maintain, and use genetically altered animal models. We also provide service on a fee for service basis to researchers in other NIH institutes. GEC can help in the design of animal models, construction of targeting constructs, ES cell manipulations, microinjections, genotyping, colony management, cryopreservation, and rederivation. Each of the six workgroups that comprise the GEC are described in sections below.
DNA Engineering Workgroup
Consultations on genetic engineering are available through GEC to provide up to date information on appropriate strategies of gene targeting and transgenic approaches in the mouse. We will also help you look into the details of the target loci and make specific recommendations on the structures of the targeting or transgenic constructs based on your goal of mutagenesis. A blueprint of the DNA construct is provided at the end of the consultation process along with estimates of time frame and cost of the entire project.
Construction of gene targeting and transgenic vectors based on the designs either provided by individual laboratories or from the consultation step with GEC. From a blueprint such as one provided through the consultation step outlined above, the GEC staff will construct the DNA construct using the recombineering technology. Currently we provide DNA construction services on all types of gene targeting vectors and transgenic DNA vectors. We also provide services on BAC clone engineering for the purposes other than generating transgenic mice.
Large scale plasmid DNA preparation is performed on the targeting vectors made by GEC, which are for the purpose of ES cell electroporation.
ES Cell Development Workgroup
Services of mouse embryonic stem (ES) cell manipulation for the purposes of generating targeted mutations or transgenics in mouse are provided through the GEC. Electroporation of mouse ES cells can be done on several lines of mouse ES cells including R1, W4 (both are Sv129) and Bruce4 (C57B6J). For each electroporation experiment, two 96-well plate-full of ES colonies are routinely picked. We generally expect ~90% of the colonies picked will successfully expand. Replica of the 96-well plates will be frozen as the source for positive clone expansion and back up. Meanwhile, one set of the 96-well plates will be continually grown under feeder-free conditions for making genomic DNA. One set of 96-well plates will be provided as cell lysates for genomic DNA preparation, usually yielding enough DNA for PCR screening, sometimes, Southern analysis too. A second set of 96-well plates is made available for those who need extra DNA for Southern verification.
This group generates transgenic mice by pronuclear microinjection of researcher-provided DNA transgene constructs into mouse embryos. This may be a service or collaboration depending on the level of initial input from unit personnel. The DNA, screening oligonucleotides and completed request form should be brought to the GEC.
We also generate chimeric mice by injecting embryonic stem cells into mouse blastocyst-stage embryos. ES cell lines may be generated by the ES Cell Unit of the GEC, or supplied by the investigator. This may be a service or collaboration, depending on the level of initial input from unit personnel.
DNA or ES cells conforming to biosafety level 1 and animal biosafety level 1 restrictions may be microinjected before a recombinant DNA registration number (RDRN) is issued, however, in these cases, the application for an RDRN, which must have been submitted to the NIH Biosafety Committee, must be attached to the submission form, and when approved, the number must be forwarded to the unit for inclusion in the official record. Similarly, DNA or ES cells may also be microinjected before an animal study protocol is approved; however, mice resulting from these procedures will not be released to investigators until their animal study protocol is approved.
This workgroup isolates genomic DNA from biopsy samples, usually mouse tail biopsies and performs PCR genotyping analyses. DNA isolation is done automatically for mice in the GEC Colony Management Unit, and on a request basis for any other mice belonging to NEI Researchers. This is done with a semiautomatic DNA extraction procedure, in batches of 96. If specific biopsy samples need to be isolated by other methods, for advanced analysis such as long PCR or Southern blot, a request can be made to have the DNA isolated manually by another method.
PCR genotyping analyses are done on DNA isolated from biopsy samples. Investigators must provide a working PCR protocol and oligonucleotide primers, and demonstrate that the protocol can detect 1 transgene copy per genome. This is done automatically for mice in the GEC Colony Management Unit, provided that essential information is provided to the unit regarding which genotype protocol(s) need to be performed.
Colony Management Workgroup
Some mice in the NEI intramural research program are maintained centrally for NEI researchers by the GEC Colony Management Workgroup. These mice are generally not actively involved in research activities, but rather are being used in breeding to generate experimental animals, or are being held for other reasons. Access to these mice by researchers is limited to escorted visits into the managed facility with one of the colony managers.
Mice in these facilities are monitored and managed by GEC colony managers. Each investigator is assigned a colony manager, who is responsible for the mice belonging to that investigator. The colony manager is responsible for overseeing all of the activities requested by the investigator. This includes setting up mating pairs, overseeing the weaning, tagging and tail biopsy of progeny, ensuring mice are delivered to investigators upon request, arranging for interfacility mouse transfers, updating database entries, submitting weekly activity reports to investigators, and communicating with investigators.
Cryopreservation & Assisted Reproduction Workgroup
This workgroup performs three major functions, 1) cryopreservation of rodent lines, 2) rederivation of rodent lines, and 3) assisted reproduction of rodent lines.
Cryopreservation: Mouse germplasm, in the form of two-celled embryos or sperm, is frozen at liquid nitrogen temperatures for long term storage of the lines. This can be done either as a safety measure for important lines currently in use, or as an efficient storage mechanism for lines no longer in active use. Upon request, a mouse line can be reconstituted from the cryopreserved germplasm, by thawing embryos and transferring into pseudopregnant females, or performing in vitro fertilization with thawed sperm.
Rederivation: Mouse lines being imported from facilities whose health status is less than that of the receiving facility's must be freed from pathogens. This is accomplished by embryo transfer from the "dirty" mice into clean mice in a clean facility. The resulting progeny of these embryo transfer procedures are then tested for pathogens, and almost always found to be pathogen-free, allowing them to enter the receiving clean facility.
Assisted reproduction: Occasionally certain lines of mice experience depressed reproductive capacity, or researchers forget to keep the line breeding until the mice are too old to breed. To help revive such lines, in vitro fertilization, using sperm from a male mouse in the troubled line and ova from young wild type female mice, is performed. If the sperm is viable, the line can be propagated in this manner.
Last Reviewed: March 2011