DNA Microinjection Services
- Introduction
- Ordering
- Turnaround Times
- Performance Guarantees
- Service Description
- Mouse Strain Considerations
- Breeding Trangenic Founders
Introduction
The injection of a DNA solution into the pronuclei of fertilized eggs is the most common method for making transgenic animals. Injection is done at the stage of development when mammalian ova have two pronuclei, one from each gamete, which will later fuse to form the diploid nucleus. 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 (around 10-15 females are used per injection day). Also, the pronuclei of mouse eggs are easy to visualize using Differential Interference Contrast (DIC) optics.
The egg donors are given two hormone injections, spaced 46-48 hours apart, to cause them to release more than the usual number of eggs and become receptive to mating – a process known as superovulation. They are then mated with fertile males and the eggs are harvested the next morning.
After injection, the eggs are transferred to the oviducts of pseudopregnant foster mothers, generated by mating females with vasectomized males so the females do not produce any fertilized embryos of their own. The offspring resulting from injected eggs may or may not carry the transgene. On average, about 15-20% of the mice we have produced from DNA microinjection have tested positive for the transgene. The mice that do carry the transgene are called founders.
Pups are genotyped using DNA extracted from a tissue biopsy (usually a small piece of tissue cut from the tip of the tail).
Ordering
To order this service, request an order form from Tom Fielder and return the signed hard copy by mail, fax, or PDF to Tom Fielder. After filling out the on-line Service Request Form, print a hard copy, have it signed by the Principle Investigator, and mail or fax it to Tom Fielder.
In addition to the Service Request Form, you must submit the following:
- A restriction digest containing approximately 50 micrograms of your plasmid, cut so as to release the transgene from the plasmid backbone;
- a gel picture of an aliquot of the digest, with the transgene band clearly identified;
- a brief description of the construct and the phenotype you hope to produce;
- Evidence of approval from your institution's Animal Care and Use Committee and Biosafety Committee for your animal use protocol and recombinant DNA protocol;
- 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 page and Construct Design page.
Turnaround Times
The minimum time required to produce transgenic founders that can be transferred to the client is about 8 weeks, once we have received a construct ready for microinjection and the necessary paperwork. This includes about 2 weeks to order and prepare the egg donors, 3 weeks gestation time, and 3 weeks from birth to weaning.
The turnaround time can be much 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
We offer two levels of service for DNA microinjection. The Basic service guarantees only that we will inject at least 100 eggs successfully (i.e., at least 100 eggs will survive the injection process and be transferred to pseudopregnant foster mothers). It does not guarantee that we will produce any pups from those eggs. The Basic service is usually completed in one day of injection.
The Premium service guarantees that we will produce at least 3 transgenic founders or 50 total pups, whichever comes first. For this service, we schedule 2 injection days to begin with and then wait to see how many pups and founders are produced before scheduling another day.
Service Description
The Basic and Premium services include:
- purchase of egg donors
- superovulation of egg donors and mating with stud males maintained by the TMF
- creation 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 ear-punch, sexing, and tail-cutting at about 2 weeks of age
- transfer of tail biopsies to the client for genotyping
- weaning, identification, and transfer or shipment of transgenic pups to the client
Mouse Strain Considerations
We offer 3 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, CB6F1. Egg donors and stud males, purchased from Harlan Sprague-Dawley, are produced by mating BALB/c females with C57BL/6NHsd males. When CB6F1 males and females are mated in our facility to produce fertilized eggs for microinjection, the eggs and resulting pups are CB6F2. CB6F1 mice are genetically identical – each pair of chromosomes consists of one BALB/c chromosome and one C57BL/6NHsd chromosome. Their CB6F2 offspring, on the other hand, while still being approximately 50% BALB/c and 50% C57BL/6NHsd, are not genetically identical, due to meiotic recombination, and will have a variety of coat colors.
The FVB/N strain (usually purchased from Charles River Laboratories) is an inbred strain, meaning all mice in the strain are genetically identical. Superovulated females produce a large number of eggs, which have large pronuclei, making them more efficient than most inbred strains for making transgenics. Information about disease susceptibilities and other characteristics of this strain can be found here.
The C57BL/6NTac strain, from Taconic, is our most expensive strain because it produces the lowest 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 we have recently switched from the C57BL/6J substrain (from The Jackson Laboratory) to the Taconic substrain. We made this change because of a significant genetic mutation in the C57BL/6J substrain that distinguishes it from all the other substrains, namely a mutation in the Nnt gene (nicotinamide nucleotide transhydrogenase), which results in a truncated protein. More details on the C57BL/6J substrain can be found online.
Other strains can be used as egg donors, but these will 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 offer only the Basic service for that strain.
Breeding Transgenic Founders
Transgenes are integrated at random into the genome. Thus, each founder will have a different site of integration. The number of copies of the transgene possessed by each founder may also be different. For these reasons, each founder should be treated as a separate line and bred independently of other founders with the same transgene.
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. However, we cannot guarantee that any expression of the transgene will be obtained.
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 more uncommon problem is loss of the transgene altogether. This may be caused by meiotic recombination, as in a double-crossover event.
If an inbred line of egg donors is used (e.g., FVB/N or C57BL/6NTac), then founders should be bred to the same background to maintain the inbred status. For founders from the hybrid CB6F1 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. 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. Some kind of 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.
