Injection of DNA into the 1-cell embryo

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 the 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 8-10 females can produce up to 200 eggs). Also, the pronuclei of mouse eggs are easy to visualize using Differential Interference Contrast (DIC) optics.

The egg donors are super-ovulated; that is they are given two hormone injections, spaced 46-48 hours apart, to cause them to release more eggs than usual. They are then mated with fertile males and the eggs are harvested the next morning.

The construct DNA is then microinjected into the pronuclei of each egg. These eggs are cultured overnight to the 2-cell stage and then transferred to the oviducts of pseudopregnant foster mothers. (Pseudopregnant females are generated by mating females with vasectomized males so the females do not produce any fertilized embryos of their own.) The mouse gestation period is 19-21 days.

Once pups are born, tissue samples will be taken for screening at approximately 7 days. These tissue samples will be sent to the Client or to a genotyping servicing company of your choice. The offspring resulting from injected eggs may or may not carry the transgene.  The mice that do carry the transgene are called founders.

Once founders are identified, the mice can either be sent to the Client for breeding or TASQ can breed the founders to produce F1 offspring (contact TASQ for conditions and charging).

DNA requirements

Researcher must supply to TASQ:

  • The linearized purified DNA construct eluted in an approved buffer
  • Concentration must be >20ng/ul and in a volume of >20ul. TASQ will dilute down to 2ng/ul at time of injection
  • A gel picture of the final DNA prep, run with several dilutions of the prep along with a DNA Mass Ladder, with the picture suitably labeled so the concentration of the prep can be estimated by comparing the staining intensities of the Mass Ladder with the construct. Alternatively a nanodrop may be used to determine DNA concentration.

It is the client’s responsibility to make sure the DNA construct has been purified correctly and the concentration determined accurately. Inaccuracies in the concentration of as little as 2-fold can have significant effects on the production of transgenic founders. TASQ performs a final dilution prior to injection to 2ng/ul. The gel picture provides TASQ with the documentation that the DNA has been purified correctly and its concentration is accurate.

Tips and protocols for preparing DNA

DNA prep

BAC DNA preparation

 

Mouse Strains Available for Injection

F1 hybrid (C57 female x CBA male) embryos are more robust with higher vigour to survive the injection and embryo transfer process. Hybrid strains have increased disease resistance and seem to cope better under stress. They have large litters as well. They are useful as hosts for tissue transplants from either the C57BL/6J or CBA/CaH/WEHI strains.

As cross is F1 X F1, resulting transgenic progeny will be F2 and of black or agouti coat colour.

C57Bl/6 Jax – Inbred embryos are less robust with lower vigour to survive the injection and embryo transfer process. C57BL/6 mice are the most widely used inbred strain throughout the world. As focus moves to transgenic and knockout mice C57BL/6 are most commonly used to produce these mice. C57BL/6 are used in a variety of different research areas. Some of these include immunology, developmental biology, diabetes and obesity, genetics and cardiovascular biology. Resulting transgenic progeny will have a black coat colour.

FVB/NJ – Inbred.  These are useful for production of transgenic mice as fertilised eggs contain large and prominent pronuclei which facilitates the microinjection of DNA. Resulting transgenic progeny will have a white coat colour. Their reproductive performance is good. 

Costs

Internal Clients: $5292/construct
External Clients: $10804.50/construct

This includes a minimum of 2 sessions of Pronuclear injections. We guarantee that 300 eggs will be microinjected for embryo transfer. Mice strains offered are F1, C57BL/6 and FVB/N strains. Costs include donor mice, superovulation, matings, culture of eggs/embryo, embryo transfers and agistment costs until progeny are weaned.

Additional Costs

  • Shipment of DNA to TASQ by Client - dependent on where the Client is.
  • Tissue sampling
  • Shipping samples to Client - dependent on where the Client is and how Clients want the samples sent. Usually sent in Australia Postpak.
  • Agistment costs once pups are weaned. Agistment is the weekly charge applied by the animal facilities for the basic care of the animal. Agistment for males (contact TASQ for agistment charges).
  • Courier costs of transgenic progeny to Client - dependent on where the Client is. Mice can be shipped with the Clients' preferred courier.  

How to start

  1. Email or phone TASQ (see Contacts page)
  2. TASQ will contact you for a consultation about DNA requirements and arrange DNA to be sent to TASQ
  3. Download and complete the registration form and return to TASQ
  4. OGTR and Animal Ethics certificates must be supplied prior to injections commencing                
  5. PN injection will be scheduled with TASQ notifying researchers of starting date.
The start date of injection will be determined by the wait list, donors required and all requirements being met by the Client.
 

Timeline

This timeline is a guide only to work performed by TASQ.

Week 1

  • Superovulation regime
  • PN Injection of DNA
  • Embryo Transfer of injected eggs

Week 4

  • Due date of birth
  • TASQ will notify researcher of birth and pup numbers

Week 5

  • Tissue samples will be collected for genotyping and sent to client or genotyping service.

Week 7

  • Progeny will be weaned. Transgenic founders can now be moved to researcher’s stocks, sent to appropriate animal facility or maintained at AIBN at additional cost to researcher.

Additional Notes

  • TASQ registration form must be completed prior to service commencement.
  • A current AEC number & certificate and OGTR number must be supplied prior to commencement of service.
  • TASQ will transfer all viable injected embryos collected and injected. TASQ can not guarantee that all embryo transfers result in live pups or transgenic founders.
  • In the situation that the guaranteed 300 eggs injected is not completed within 2 microinjection sessions, the Client will be notified and another round of injections will be scheduled.
  • If the Client is responsible for genotyping, it is asked that the Client return to TASQ the full list of samples sent with a corresponding result and action.
  • TASQ will not cull any wildtype progeny until a written request is received from the Client.

FAQ

1. What if there are no births or founders from the injection sessions?

TASQ can not guarantee that 100% of embryo transfers will result in live births. Over the injection sessions, different staff members perform the egg collections, egg culture, micro- injections and embryo transfer. A control embryo transfer of uninjected embryos will be performed where possible alongside each transfer of injected embryos. If there are no births TASQ will request a new DNA prep be made by the client and another session will be arranged at cost to client. TASQ can purify the new prep if requested.

2. How important is the DNA prep to the success of the transgenic production project?

The success rate of transgenic production is heavily influenced on the quality of the prep supplied to TASQ. A pure and clean DNA prep will greatly increase the chances of transgenic founders produced compared to the same prep of lesser quality. This is why TASQ asks that the DNA preps are produced with proven protocols (see above section Tips and Protocols for Preparing DNA for links to how to prep the DNA prior to injection). If no founders are identified from the first round of injection sessions, TASQ will ask for a new prep and consult with client on the DNA prep protocol.

3. What are the variables in the production of transgenics?

  • As the integration of the construct is random, clients should be aware that the position of integration and number of copies may differ between founders so each founder will need to be assessed for expression and function of the transgene.
  • Late integration may also occur which results in a mosaic founder. This means that the usual mendelian principles of breeding may not occur, with a founder having a low percentage of transmission.
  • There is no guarantee that founders integrating the transgene will show expression and a phenotype. Expression levels also do not mirror copy number integrated.

4. When is the Client notified of progress?

TASQ will notify the Client:

  • when all forms, certificates and information is received by TASQ
  • when the microinjection sessions have been scheduled
  • when the microinjection and embryo transfers have been completed
  • when resulting progeny are born
  • when the transgenic founders are ready to be released to the Client. 
Pronuclear Injection References
 
  • Brinster, R.L. et al (1985) Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs. Proc. Natl. Acad. Sci. 82, 4438-4442.
  • Gordon, J.W. et al (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proc. Natl. Acad. Sci. 77, 7380-7384.
  • Jaenisch, R. (1988) Transgenic Animals. Science. 240, 1468-1474.