Transgenic Mouse: Pronuclear Injection

How do you make a transgenic mouse?

Here's a general overview of the process, including the responsibilities of the client and the resource.

  • New clients consult with manager, juzhao@ad.ucsd.edu
  • Client creates transgene plasmid
  • Client cuts transgene from plasmid and purifies DNA according to the recommended protocol
  • Core prepares pronuclear-stage embryos
  • Core injects transgene into embryos
  • Core implants embryos into foster females
  • Client screens resulting pups for transgene

For a more detailed description, please see the Practical Guide below.

A practical guide for making transgenic mice

Introduction

Targeting of transgene expression to different cell types is achieved with the use of fusion gene constructs in which a coding sequence of interest is placed downstream of a cell-specific promoter and upstream of a polyadenylation [p(A)] signal sequence. The fusion gene construct is then microinjected into one of the pronuclei of fertilized mouse eggs, which are subsequently implanted in the oviduct of pseudopregnant recipient mice. Progeny are analyzed for transgene integration and positive mice subsequently mated, their offspring screened for transgene expression and, where positive, used to develop transgenic lines with stable integration and expression of the transgene. The procedures for the production of transgenic mice are technically demanding and the success of this approach can be influenced by a number of variables--both technical and biological. Key points to be addressed here correspond to the circled numerals in the figure.

Transgene schematic: 1. Fusion gene construct, 2. Superovulated female mouse, 3. Microinjection, 4. Birth of transgenics, 5. DNA analysis, 6. Breeding positives to establish transgenic lines

1. The Fusion Gene Construct (Investigator Responsibility)

The concentration, size and purity of the DNA to be microinjected are important factors for the successful production of transgenic mice. For optimal efficiency of DNA integration and egg survival, DNA is normally microinjected at 1-2 µg/mL in injection buffer. This applies whether we are injecting a single fragment or co-injecting two or more fragments. The size of the DNA determines the molar concentration and therefore the number of copies of the gene per injection sample. For example, injection of 2 picoliters of a 2 Kb fragment at 1µg/mL, represents about 1,000 copies. An ideal fragment size is between 2 Kb and 20 Kb.To have a fragment much under 2 Kb is not recommended because, empirically, it usually does not integrate very efficiently. We have successfully injected fragments as large as 40-50 Kb. The larger the DNA or the more fragments that are in the injection sample, the lower the final copy number of individual genes becomes in that sample. This usually does not matter, but can sometimes result in lower integration efficiencies. Offsetting this reduction by injecting larger amounts of DNA is not recommended because the number of injected embryos surviving to produce live pups diminishes.

Another important factor is the purity of the DNA. It has been well established that plasmid DNA sequences can dramatically influence the function of foreign genes, therefore all vector sequences should be removed from the cloned gene to be microinjected, if optimal expression of that gene is desired.

The quality and purity of the isolated fusion gene DNA fragment is a critical determinant in efficiency of chromosomal integration and egg survival. DNA should be free of damage, and chemical, microbial and particulate contamination. We have a protocol in the facility that we strongly recommend for DNA preparation, which will maximize your chances of success. DNA for microinjection is finally resuspended in microinjection buffer (7.5 mM Tris, 0.15 mM EDTA pH7.4) and an accurate assessment of the concentration is made, prior to delivery of the DNA to the transgenic core. Within the facility we shall confirm the concentration of your DNA by gel analysis, and will dilute it appropriately immediately prior to injection. We inject at a concentration of 1-2 µg/mL, a few pico liters of solution into each embryo. You will need a minimum of 200-300 ng of DNA in your final preparation.

2. Collection of Fertilized Embryos from Superovulated Female Mice (Core Responsibility)

Fertilized ova for the microinjection of DNA are obtained from female mice that are sired with stud males. To increase the yield and quality of eggs, female mice are superovulated with gonadotrophins. Fertilized embryos are harvested after dissection of the oviduct from newly plugged mice. In our facility, the procurement and subsequent handling of embryos is relatively straightforward. There are, however, biological variables that can dramatically influence the quantity and quality of eggs for microinjection. In particular, the mouse strain plays a key role. Hybrid strains have been most commonly used for their desirable reproductive characteristics and enhanced egg quality. Even with hybrid strains, mating efficiency, egg yields and quality can occasionally vary unpredictably. Efficiencies decline significantly when using many common inbred strains and in some cases (e.g. SJL or BALB/c ) are so low as to preclude their use for this purpose. Currently, in the Cancer Center Core, we work primarily with the hybrid strain CB6. This strain is an F1 hybrid between a BalbC and C57 Bl6 mouse. The embryos that we microinject are the F2 products of mating the F1’s.

Occasionally, we can accommodate an inbred strain such as a C57Bl6 or another hybrid strain if CB6 will not meet your needs, but we have severe space constraints which limits what we can offer in this regard. Also of note is that the cost charged for the use of inbred mice is somewhat higher than for hybrid strains, because more time and effort is involved in the production of inbred transgenic lines.

3. Microinjection of DNA into Fertilized Embryos (Core Responsibility)

Microinjection of DNA into Fertilized Embryos

The most technically sophisticated and demanding step in the development of transgenic mice is the microinjection of the DNA into the pronucleus of a fertilized ovum. This step is critical and requires considerable technical proficiency and extensive training to master the microinjection procedure. Once this skill is acquired however, technical variability and errors become minimal. The routine survival of microinjected embryos is between 80-90% in our facility. Groups of 20 embryos, once injected, are reimplanted into the oviduct of pseudopregnant female recipients. These are females mated with appropriate timing to vasectomized male mice. They will give birth 19-20 days after implantation. We offer two basic microinjection schedules--a full injection series (~280 embryos implanted) or a half injection series (~140 embryos implanted). Fewer mice will be generated from the half series, but you will save money! Half jobs are recommended usually only for experienced investigators who are routinely generating multiple transgenic lines per construct. The number of offspring resulting from the implanted embryos will vary, depending not only on the number of implants but also on the quality and quantity of the DNA injected and to some extent, on biological variations out of our control (e.g. mothers eating pups, etc.) Typically, however, from a full injection series of embryos on a hybrid background one might expect 50-60 pups, of which the actual number that will be transgenic should be in the range of 10%-40%. It cannot be stressed enough that a major determinant for the final yield of transgenic mice is the initial concentration and purity of the microinjected DNA. It is well worth the time and effort up front to give us premium DNA in order to avoid wasted time and money and much frustration later on.

4. Analysis of Tail DNA for Transgene (Investigator Responsibility)

One to two weeks after birth, lactating mothers with litters will be shipped out of the transgenic facility to the investigators own mouse rooms. Litters need to be weaned at 3-4 weeks of age and transgene integration can be assessed by tail tissue analysis. It is imperative that you have a probe that is unique and specific enough to detect integrated DNA even in low copy numbers. Occasionally, mosaic mice occur, where the DNA integrated at a two cell or later stage embryo. In this case, you will have less than a single copy per cell of your gene present for detection. There is no guarantee of getting specific numbers of transgenic mice. However, our track record shows that expectations of the number of transgenic lines obtained from a full injection job into a hybrid strain (with DNA of the correct concentration and purity) would be between five and ten. Integration efficiencies into inbred strains such as B6 will be about the same, but there will be fewer pups born in the first place. If you have had two or more fragments co-injected, the number of transgenic mice with both fragments versus one or the other will be a matter of luck.

5. Mating Transgenic Mice (Investigator Responsibility)

At around 6 weeks of age, matings should be set up between your transgenic positive mice and wild-type mice. Transgenic females are mated one-on-one with a male, and transgenic males can be mated with two or three females at a time. Most times, the transgene is transmitted in a Mendelian fashion. If you have a mosaic mouse, transmission may be much sparser than Mendelian, and you may have to breed several litters to find your transgene. Once you have a positive offspring, breeding from that one should be Mendelian. At other times (rarely), you may have more than one integration site, in which case your transgene will be transmitted in greater than Mendelian numbers. Very rarely, the transgene is not in the germline and is not transmitted. Expression of the transmitted transgene is obviously construct and to some extent, integration site dependent.

A Timeline for Transgenes

Once the targeting construct is made and delivered, the transgenic service is on a first come first served basis. For a standard hybrid strain, there is usually only a wait of 1-2 weeks before we can inject the construct. Mice will follow five weeks later.

Recently, some investigators have been using transient transgenics to obtain preliminary information. Here, there is no breeding because females with potential transgenic pups in utero are sacrificed during gestation. If this application is suited to your experiments, not having to breed transgenic lines can obviously save both time and money.