Jordan Kitchen and Dr. Sandra Burnett, Micro and Molecular Biology
Transgenic mice are used to create animal models and are typically generated by the addition of transgenes (4-14 Kb pairs) or artificial chromosomes (150-300 Kb) to the pronucleus of a fertilized mouse egg [1][2]. Because of the size of chromosomes, using them to produce transgenic mice can be very problematic. Current methods for inserting transgenes include delivery through a microinjection device into the pronucleus. Transgenic mice are then created by transplantation of injected eggs into a surrogate female [3]. Larger DNA or yeast artificial chromosomes (YAC) due to the increased amount of genetic material could be used to express an overall greater change in the cell. However, current methods of injection are problematic because the YAC DNA is too large to fit through the microinjection needle without suffering substantial damage – damage is caused by brute forces and a narrow needle that result in shearing of the DNA [3]. For the past year I have been working with a research team developing a new injection device: the nanoinjector. Part of this research was focused on different methodologies and concentrations required to successfully inject the YAC YRT2 and YRT3 (tyrosinase genes) into a transgenic animal.
Preliminary testing showed that a concentration of 5-7 ng/ul of YAC YRT 2 was toxic to the cell. One hundred of the injected eggs that had progressed to the 2-cell stage were implanted into six surrogate mothers. Three gained weight, and two of these mothers’ pregnancies resulted in pups (14 from one mother and 2 still-born pups from a Csection performed on the other mother). Expression of the chromosome was analyzed by a color change in the pups. Pups with the chromosome incorporated would no longer be white in coat color because both YAC YRT2 and YRT3 have the required genes to correct for a mutation dealing with coat color. None of the pups expressed the YAC YRT2 transgene. The most significant finding for the progeny of the 5-7 ng/ul concentration of YAC YRT2 was that upon dissection to harvest the decidua (uterine lining), we found that three embryos were being reabsorbed. This led us to conclude that a concentration of 5-7 ng/ul is toxic and leads to abortion.
We then modified the concentration to 0.5-0.7 ng/ul. Thirty-six 2-cell eggs were implanted into two surrogate mothers. One of these mice gained weight. This mouse then produced ten offspring, all of which were negative for expression of YAC YRT2. We then changed the concentration to 0.05-0.07ng/ul. At this concentration, ninetythree 2-cell eggs were implanted into six surrogate mothers. All but one of these mothers showed weight gain. The nineteen resulting progeny were from only four mothers because one mother aborted. Three of the mice had five offspring each and the other had only four. Because we were not able to get the chromosome to successfully incorporate, we thought that maybe not enough complete copies of the chromosomes were being injected. To eliminate this variable, we repeatedly tried the concentration of 1.4-2.0 ng/ul as was suggested in literature and through collaboration with other researchers. A total of 258 eggs at the 2-cell stage were injected into eleven mice. Six of these mice gained weight resulting in offspring without expression.
When things were still not working out, I went to Madrid, Spain and learned how to purify and make the chromosomes myself. I did an internship at the Centro Nacional de Bioltecnologia in the lab of Dr. Montoliu. They had supplied us with the chromosomes. Here I learned to prepare YACs and prepared some samples of YAC YRT2 and YRT3 to use in the studies back in the states. The difference between the two YACs is that YAC YRT3 is smaller but still contains the necessary coding regions. My preparations of the YACs were microinjected into eggs that were then implanted into surrogate mothers. The technicians said that the YACs were very pure and easy to inject. The YACs yielded 80% integration in the pups. Normal integration with microinjection is around 1-6%, usually residing at 1%, signifying that the new preparations worked.
After I returned to the states, we set up a comparison study with the University of Utah Transgenic and Gene Targeting Mouse Core. We nanoinjected 2 ng/ul and microinjected 1ng/ul of YAC YRT3. I decided to shift to the smaller sized YAC to try and get more copies integrated. During collaboration at the International Transgenics Technology Society 2011 conference, the poor quality of the donors was decided to be responsible for the low integration and survival rates. In this study, 195 nanoinjected 2-cell embryos were placed into ten surrogate mothers and forty-one microinjected 2- cell embryos were placed into two surrogate mothers. One of the nanoinjected mothers died containing a pup. The others yielded fifty-three pups. Microinjection yielded only eleven pups between the two mothers. None of the pups expressed the color change, which would signify the integration of the YAC. This was interesting because both the micro and nanoinjection did not yield any transgenic mice. We think this could be due to damage to the YAC during shipping and could explain the negative results previously. However, it is also possible that we could have just not had a large enough population size, since it is typical to only get 1% of pups to express the gene of interest. This is a work in progress and is still being researched. Currently I am developing different methods to tests for integration and have five PCR primers to test whether partial integration has occurred. Other ideas for further research are to purify the YACs here so they do not have to be shipped and to increase the experimental size.
References:
- Ohtsuka M, Ogiwara S, Miura H, Mizautani A, Warita T, Sato M, Imai K, Hozumi K, Sato T, Tanaka M, Kimura M, Inoko H, Pronuclear injection-based mouse targeted transgenesis for reproducible and highly efficient transgenesis expression, Nucleic Acids Res. 2010 Sep 29. PMID 20990997
- Pedro N. Moreira, Julio Pozueta, Patricia Giraldo, Alfonso Gutierrez-Adan, and Lluis Montoliu, YAC Protocols: Second Edition, Generation of Yeast Artificial Chromosome Transgenic Mice by Intracytoplasmic Sperm Injection, Methods in Molecular biology, vol. 349
- Patricia Giraldo and Lluis Montoliu, Size matters: use of YACs, BACs and PACs in transgenic animals, Transgenic Research 10: 83103, 2001
Special Thanks to:
Brigham Young University MAFIA Research Laboratory and Compliant Mechanisms Research Laboratory, Technicians of the University of Utah Transgenic and Gene Targeting Mouse Core, Centro Nacional de Biotecnologia Laboratorio de Dr. Lluis Montoliu, and for NanoInjection Technologies, LL