Lisa Higgenbotham and Dr. Robert Seegmiller, Physiology and Developmental Biology

Microinjection is a technique used to introduce DNA into the genome of an animal at the embryo stage. It is an effective way to insert replacement, or rescue, copies of a gene that the embryo may be lacking. In this study, I prepared purified copies of the mouse Col2a1 gene and microinjected them into the pronuclei of single-cell mouse embryos. Col2a1 encodes type II collagen, a major component of cartilage. Mice carrying one or two mutant alleles of Col2a1 are characterized by developmental abnormalities, degenerative joint disease, and even death. This phenotype is observed in Disproportionate micromelia (Dmm) mice, which harbor a mutation in the C-propeptide region of the Col2a1 gene. I hypothesized that introducing functional copies of this gene would override the effects of the mutant alleles and generate a strain of Dmm mice with attenuated manifestations of the mutant phenotype.

The majority of time for this project was spent learning and practicing the techniques, specifically dissection, handling mouse embryos, microinjection, and embryo transfer surgery. The last of these was particularly difficult to master. Surgery involved administering anesthesia, making two small incisions, locating the opening (ostium) of the oviduct, and mouth-pipetting embryos into the opening with a glass pipette. All these steps had to be done within about five minutes, followed by suturing, administration of analgesic, and monitored recovery. It was truly a challenging, but remarkable experience to single-handedly perform surgery on a living mammal, and then see it recover.

After the extended training period, I had five attempts to carry out the complete process. To outline the process briefly, first I prepared the Col2a1 transgenic DNA—isolated, purified, and diluted it to a precise concentration. Meanwhile I gave hormone injections to young female mice, and set up matings. From these females, I surgically removed <1 day-old embryos from the oviducts. Next I washed the embryos (sometimes over 70 at a time) with buffers and enzymes, using a mouth pipette to transfer the embryos from one wash to another. Once that was done, I set up the microinjection apparatus by filling the tubing with oil, changing the tiny needle, and loading the needle with DNA. Then I placed embryos on a slide under the microinjection microscope, and individually located a pronucleus in each embryo, and poked through the outer layers of each cell. At this point, ideally the transgenic DNA would flow into the mouse embryo. However, I encountered trouble getting the DNA to pass through the needles. One possible explanation is that the large size of the Col2a1 construct blocked flow. Air bubbles may also have interfered. Because of this complication, it was impossible for the microinjection to yield a transgenic embryo. Realizing this, I performed a side-study to confirm that the other steps of the procedure weren’t also problematic. I demonstrated that embryos survive the preparatory steps, and that embryos that were poked with the microinjection needle survived and grew to at least the two-cell stage at a 67% rate, compared with an 80% rate for non-poked control embryos. The surgical procedures that follow microinjection proceeded with variable success, but none of the attempts resulted in pregnancies. There are a few possible explanations for this: embryos were reabsorbed by the uterus; adverse reaction to anesthesia; infection was introduced during surgery; and chance—if more trials had been performed, eventually one would work. Even though the surgeries described above did not yield any viable, and possibly transgenic, pups, I prepared a way to test for the transgene in the future. I designed DNA primers that amplify a 550 bp portion of the transgene. I tested these primers on purified transgene and on DNA samples from mice in the Dmm colony. PCR was successful, and restriction digest allowed me to differentiate between transgenic and nontransgenic samples. Although the trials I performed did not generate a transgenic mouse, I am fairly confident that one could be made in the future. Given proper training and resources, it just takes repetition before all the steps work out together. Besides a few more months for trials, a few things that would have helped me include a partner to load and hold the transfer pipette during surgeries, and injection needles with a slightly wider diameter to facilitate DNA flow. With these recommendations put into action, I believe future microinjectionists at BYU will achieve the goal. The strongest conclusion that can be drawn from this project is that microinjection is a complicated procedure, and it demands large amounts of time, practice, and troubleshooting. It is no wonder that research institutions typically reserve facilities and technicians solely for the production of transgenic mice. Nevertheless, for a student interested in learning a wide range of skills, from basic molecular biology to live surgery, a project like this provides outstanding exposure and experience. During the course of this project, I prepared a 33-page manual detailing the procedures involved in the complete microinjection process. This manual is now included in my Honors thesis, which was successfully defended on May 24, 2006. Additionally, I made a slide-show movie to accompany the manual. The movie features a walk through the techniques, and I have shown it to many groups and individuals, both scientific and not. The general response has been one of increased interest and enthusiasm toward research. Most importantly, this project has opened up new opportunities. For one thing, I trained an incoming PhD student in all of the procedures. This student is now incorporating microinjection into her doctoral research at BYU. She is also making progress to resolve the DNA flow through the needles. As for myself, I am now a PhD student at Cambridge University in England, in a partnership program with the National Institutes of Health. The experience I gained through my ORCA project played a significant role in my decision to apply to graduate school, and in my admission to an outstanding program. I am continuing my work with mouse models of human diseases, and the skills described above are now helping me toward my PhD. To say that my ORCA project was worthwhile would be an understatement. It made all the difference.