DNA Microinjection Services


Since 1980 transgenic mice have routinely been generated by injecting a solution of DNA into a pronucleus of a fertilized egg. In a proportion of injected eggs, the DNA becomes integrated into one of the chromosomes. Cells descending from this fertilized egg, including germ cells, can contain the transgene DNA. Mice with the transgene can be bred and analyzed to investigate the effect of the transgene on mouse development, reproductiion or homeostasis.

Mice are particularly suitable for this procedure because it is easy to collect large numbers of fertilized eggs from a relatively small number of animals. To produce eggs, the egg donors are given two hormone injections spaced 46-48 hours apart.  This process (termed superovulation) causes them to release more than the usual number of eggs and become receptive to mating. The superovulated female mice are mated with fertile male mice and the eggs are harvested the next morning.

Microinjection of DNA is performed shortly after fertilization. The pronuclei of mouse eggs are readily visualized using Differential Interference Contrast (DIC) optics (see image to right).  Fertilized eggs have two pronuclei, one from each gamete. The injected DNA can integrate into chromosomes in the injected pronucleus during chromosome replication.  After the DNA in each pronucleus has duplicated, the pronuclear envelopes break down, the duplicated chromosomes align on the metaphase plate and the fertilized oocyte divides to form a two-cell stage mouse embryo.

Following injection with DNA, surviving fertilized eggs are surgically transferred to the oviducts of pseudopregnant mice.  Pseudopregnant mice are produced by mating fertile females with vasectomized males.  Because the males are sterile, the females do not produce any fertilized embryos of their own.  However, the process of mating stimulates their reproductive tract to support development of fertilized eggs following surgical transfer.  If the injected DNA has integrated into a chromosome, the offspring resulting from transfer of the injected eggs should contain the transgene in some or all of the cells in their bodies.

After mouse pups are born a small tissue biopsy is procured and DNA is extracted and analyzed for the presence of the transgene. On average, about 15-20% of the mice we have produced from DNA microinjection have tested positive for the transgene. The initial mice that carry the transgene are called 'founder' (or F0) transgenic animals.  The F0 mice are subsequently mated with wild type mice to identify F1 offspring containing the transgene and new lines (strains) of transgenic mice are established.

Although popular, this method of producing transgenic mice has some disadvantages (see the discussion below on breeding transgenic founders). To overcome these, we now offer the capability of inserting single-copy transgenes into the ROSA26 and H11 loci.  In addition to providing more consistent gene expression between animals, this method requires less effort on the part of the investigator. Of equal importance, fewer animals are required to perform experiments using this method.  For more information see the description of our Targeted Transgenesis service.


Contact the TMF for a Service Request Form. The completed form must be signed by the Principal Investigator.  Signed forms can be e-mailed to the TMF as a scanned image.

In addition to the Service Request Form, the following materials are required:

  1. A restriction digest containing approximately 50 micrograms of your plasmid, cut so as to release the transgene from the plasmid backbone.
  2. A photo of an aliquot of the digest analyzed by agarose gel electrophoresis, with the transgene band clearly identified.
  3. A brief description of the construct and the phenotype you hope to produce.
  4. Evidence of approval from your institution's Animal Care and Use Committee and Biosafety Committee for your animal use protocol and recombinant DNA protocol.
  5. For off-campus clients, a Material Transfer Agreement, including name(s) of your transgene(s), signed in duplicate.

Tips and protocols for preparing and testing your DNA construct can be found on our DNA Preparation and Construct Design pages.

Turnaround Times

Following receipt of a construct ready for microinjection along with the necessary paperwork, the minimum time required to produce transgenic founders that can be transferred to the client is about 8 weeks.  This includes about 2 weeks to order and prepare the egg donors, 3 weeks gestation time, and 3 weeks from birth to weaning.

Please note - the turnaround time can be longer, either because of previously scheduled orders or because of a failure to produce founders from the first injection. Delays may also be caused by technical difficulties in identifying transgenic founders, which is generally the responsibility of the client. In order to minimize turnaround times, we recommend optimizing and testing your genotyping protocol well in advance of pups being born. For PCR assays, test the specificity and sensitivity by diluting your transgene into genomic DNA from mouse tails (available from the TMF), down to a concentration equivalent to a single-copy gene. For a transgene that is 3 kb in length, this would be equivalent to 0.1 picogram of transgene in 100 nanograms of genomic DNA.

Performance Guarantees 

Our Standard DNA Microinjection Service guarantees the production of 3 transgenic founders or 50 total pups, whichever comes first. For this service, we first perform two days of DNA injection.  After tissue bopsies from the resulting offspring are assayed for the presence of the transgene, additional days of microinjection are scheduled as needed.

Our One-Day DNA Microinjection Service is, as the name implies, designed to be finished in one day of injection.  This service is offered for the B6SJLF2/J and the C57BL/6NJ strain, although low yields of eggs and pups are often obtained from the C57BL/6NJ strain.  While we do not guarantee to produce any pups or founders from this service, if one day of injection fails to produce at least 1 transgenic founder and the egg yield for that day was unusually low (more than one standard deviation below the mean for the last 25 injections on the same background), we wil repeat it once at no additional charge.

Potential users of either service should be aware that presence of a transgene does not guarantee expression of the transgene.

Service Description

Both the Standard and One-Day services include:

  • Purchase of egg donors.
  • Superovulation or hyperovulation of egg donors and mating with stud males.
  • Production of pseudopregnant foster mothers by mating outbred ICR females with vasectomized males and checking for vaginal plugs the morning of injection.
  • Harvesting of eggs from euthanized donors.
  • Dilution of the client’s transgene DNA with injection buffer to a concentration of 2 ng/ul and injection of this solution into one pronucleus of each fertilized egg.
  • Transfer of all eggs that survive the injection process into the oviducts of pseudopregnant foster mothers.
  • Monitoring of foster mothers before and after birth of pups.
  • Marking pups in each litter by distal toe-clipping, sexing, and (if required) tail-cutting at about ten days of age.
  • Transfer of toe or tail biopsies to the client for genotyping.
  • Weaning, identification, and transfer or shipment of transgenic pups to the client.

Which Mouse Strain Should I Use ?

We offer 2 strains of egg donors on a routine basis. The service fee varies by strain because of differences in egg and pup yields among these strains.

The strain with the highest yields is the hybrid strain, B6SJLF2/J. Egg donors and stud males are purchased from the Jackson Lab (Jax), where they are produced by mating C57BL/6J females with SJL/J males. When B6SJLF1/J males and females are mated in our facility to produce fertilized eggs for microinjection, the eggs and resulting pups are referred to as B6SJLF2. B6SJLF1/J mice are genetically identical - each pair of chromosomes consists of one C57BL/6J chromosome and one SJL/J chromosome. In contrast, their B6SJLF2 offspring, while still being approximately 50% C57BL/6J and 50% SJL/J, are not genetically identical due to meiotic recombination, and will have a variety of coat colors.

The C57BL/6NJ strain is more expensive because it produces a lower egg and pup yields. However, its rate of transgenesis (percentage of pups that are transgenic) is about the same as the other strains we routinely offer (around 15-20% of all pups are transgenic). Please note that unlike the C57BL/6J substrain, C57BL/6NJ are wild type for the Nnt gene (nicotinamide nucleotide transhydrogenase). More details on the C57BL/6J substrain can be found online.

While other strains can be used as egg donors, these usually require additional fees. Some lines are very poor donors and should be avoided if possible (e.g., BALB/c and DBA/2J). If no data is available on the suitability of a given strain for use as egg donors, or if it is known to be problematic, we can only offer the One-Day service.

Breeding Transgenic Founders

When microinjected into pronuclei, transgenes are integrated at random into the genome. Thus, each founder will have a different site(s) of integration. The number of copies of the transgene possessed by each founder may also be different and there may be more than one site of integration within the genome. Also, some founders may be mosaic for the transgene. For these reasons, each founder should be treated as a separate line and bred independently of other founders carrying the same transgene (i.e., don't mate founders with each other).   

Offspring must be obtained from each founder to test for transmission and expression of the transgene. Due to position effects and different copy numbers, each founder line can have a different level of expression.  Indeed, we cannot guarantee that any expression of the transgene will be obtained.  Use of insulator sequences in a transgene construct can help prevent such ocurrences.

The transgene will not necessarily be transmitted in a Mendelian fashion by a given founder. Founders can be mosaic for the transgene, if integration occurs after the first cell division. Mosaicism can result in a frequency of inheritance of less than 50% in the first generation offspring. In some founders, the transgene may integrate into more than one locus, resulting in a frequency of inheritance of more than 50%. In this case, the expression levels among the first generation offspring may vary, depending on which integration site they inherit.

A less common problem is loss of the transgene altogether, which may be caused by meiotic recombination.  Occasionally, transgene expression can be shut off in subsequent generations due to methylation, which may be connected with the fact that most transgenes are inserted as head-to-tail concatemers containing multiple copies of the transgene.  We now offer other methods of making transgenic mice, using homologous recombination at the ROSA26 and H11 loci, that eliminates the problems associated with randomly inserted transgenes.  See our Targeted Transgenesis service.

If an inbred line of egg donors is used (i.e. C57BL/6NJ), then founders should be bred to the same background to maintain the inbred status. For founders from the hybrid B6SJLF2/J strain, the client must decide whether to backcross to an inbred line, or to maintain a mixed background.

Mice that have the transgene on one chromosome are termed "hemizygous" because they do not have a corresponding allele on the other chromosome. In many cases it is possible to produce homozygous transgenics, and these may have a higher level of expression than the corresponding hemizygous mice, but distinguishing homozygotes from hemizygotes can be difficult. A quantitative genotyping assay must be used (e.g., quantitative PCR or Southern blotting). Alternatively, suspected homozygotes can be crossed to wildtype mice and all offspring tested to see if they are hemizygous.

Finally, investigators should be aware that in approximately 10% of cases, integration of the transgene is associated with changes in genome structure (i.e. deletions, insertions, inversions) that can result in a recessive mutation.  The mechanism underlying this is not clear, but may involve physical damage to compacted chromatin by the micoinjection needle during injection of the DNA transgene.

Current pricing for these services: