Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.
In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.
For nearly 25 years, the creation of genetically targeted mice has been a slow process, often hampered by a bottleneck at the blastocysts ES cell microinjection stage for the generation of germline transmitting chimeras. Recent advancements, including CRISPR/Cas91,2 targeting in ES cells and high-throughput ES cell generation consortiums like KOMP, have improved the generation/availability of genetically modified ES cells. However, the generation of germline chimeras remains a bottleneck in creating genetically modified mice from these ES cells3,4. High-throughput ES cell generation projects have been plagued by high variability in ES cell quality, which is critical for successful creation of germline chimeras. Additionally, some of the commonly utilized ES cell lines are known for high aneuploidy, and difficult production of germline competent chimeras5.
Many methods have been developed for creating mice with either a higher chimera, or completely ES cell derived mice6,7,8,9,10. While each of these systems has its own merits, they also have their shortcomings. The generation of tetraploid chimeras, while generating 100% ES cell derived mice, is inefficient, and generally limited to outbred lines 5,6. Newer methods of injecting morula can yield high percentage chimeras, approaching 100%, but generally require significant additional equipment (lasers or piezos) and training10,11. In laboratories where these techniques are already in place, this new methodology may not be necessary.
This study's objective was to find a method that uses common techniques and readily available equipment to increase the rate of chimera generation, increasing the chance of germline transmission of the mutant allele to establish the subsequent mouse colony.
All operations were done as part of the normal operation of the NIEHS Knock Out Mouse Core facility. All mice were kept on a 12:12 h light:dark cycle, and were feed a diet of NIH-31 and water ad libitum. All experiments were performed in accordance with the Animal Care and Use Committee of the National Institute of Environmental Health Sciences.
1. Animal Preparation
2. ES Cell Preparation
3. Embryo Collection
4. Embryo Injection
5. Transfer of eEmbryos
Treating cell-line as the random effect, a mixed effects linear model was used to analyze the data in the software (e.g. SAS (version 9.2)). Each analysis controlled for the number of embryos transferred and the cell line. The second analysis also controlled for the number of pups that survived.
For this study, each ES Cell line and clone was injected both with traditional and TICM techniques, alternating between the two methods after each embryo. After injection, embryos were segregated on the dish and grouped by transfer, generally between 12 and 15 embryos per recipient female. Embryos that did not have a clearly visible blastocoel, lacked a Zona pellucida, or where the microinjected ES cells failed to enter the blastocoel, were eliminated from this experiment.
The first data examined was pregnancy rate. As shown in Figure 3, pregnancy rates were unaltered in the TICM group from the control group. Additionally, an equal proportion of pups survived to weaning (P = 0.50, Figure 4). Most importantly, while the number of mice at weaning remains unchanged, the number of pups displaying coat color chimerism increased from 31% to 50% for the TICM group (p = 0.005, Figure 5). The degree of coat color chimerism was considered too subjective to be evaluated in this study, and not truly relevant to germline chimerism.
Figure 1: Specialized Equipment. (Left) Injection microscope setup, including (A) Micro Manipulators and (B) Cell Tram Air. (Right) Mouth pipette apparatus, with (C) drawn glass pipette, (D) 1000µL pipette tip, (E) tubing, (F) Filter, and (G) Syringe barrel. Please click here to view a larger version of this figure.
Figure 2: Example of blastocyst embryo injection. (A, B) Traditional injection method, where the injection needle enters opposite of the ICM, avoiding any interaction with the ICM. (C, D) TICM method, where the injection needle enters directly through the ICM in to the blastocoel. Please click here to view a larger version of this figure.
Figure 3: Pregnancy rate. The pregnancy rate showed no significant difference between traditional (39/50) and TICM (64/76). Averages plus standard error of the mean (SEM). Please click here to view a larger version of this figure.
Figure 4: Survival rate. The survival rate is the number of pups that survived to weaning, where there was a litter to wean. Pups that did not survive to weaning, and litters where no pups survived to weaning, were not included in this data. Averages plus standard error of the mean (SEM). Please click here to view a larger version of this figure.
Figure 5: Chimerism rate. This data only represents the number if chimeras produced, and does not address the quality of chimerism. In the traditional group, 31% (34/110) of the offspring showed chimerism, where the TICM group had an average of 50% (79/158). Average plus standard error of the mean (SEM). Please click here to view a larger version of this figure.
It has long been thought that any disturbance of the ICM could lead to mortality, in fact, current blastocyst microinjection protocols still warn of this12,13. What we have shown here is that microinjection through the ICM is not detrimental to the embryo, and increases the yield of chimeras.
The exact mechanism for this effect has not been identified. However, the mechanical disruption of the ICM, and the accompanying hypoblast, should increase the incidence of ES cell integration into the newly formed ICM following microinjection. Recent work in the field of iPSCs has shown that mechanical stimulation helps in the reprogramming of human iPSC14. If mechanical manipulation has a similar effect in the ICM of the mouse embryo, it could have the effect of returning cells of the ICM which had potentially started to differentiate, back to a pluripotent state, removing a potential 'head start' of the blastocyst donor derived cells in forming the embryonic tissue. Lastly, it is possible that the injection through the ICM results in the death of an unknown number of cells in the ICM, lowering the level of competition for the microinjected genetically modified ES cells.
The primary benefits of this method modification are that it requires no new learning, troubleshooting, or equipment. All the techniques carry over from standard blastocyst injection. The most critical steps also remain unchanged; proper and timely collection of embryos, and proper culture conditions for the embryos before, during, and after injection. When using the non-surgical embryo transfer technique, it does become more critical to select proper recipient mice. With surgical transfers, the surgeon had the opportunity to examine the mouse to verify proper reproductive state, where with non-surgical transfers, this is not possible. Mice should be of prime reproductive age (6-9 weeks) and only mice with visible plugs should be used. The most important aspect of this technique, and all ES cell injections, is the quality of the ES cells.
When dealing with high quality, robust cell lines, this technique may yield only minimal differences, but with suboptimal ES cell lines or difficult to work with lines, any, even minor improvements, can be significant and make the difference between project success and failure. It is also of note, that over the course of this study, several commercially available ES cell lines generated germline chimeras, and nearly all of the commercial ES lines that generated germline chimeras were TICM injections. Here we have shown that a simple change in technique can result in a significant improvement in productivity, without incurring any additional costs for new equipment, training, or increasing the use of animals.
The authors have nothing to disclose.
This research was supported [in part] by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. Special thanks to Shyamal Peddada for statistical analysis. We also wish to thank Humphrey Yao, Gary Bird, and Yuki Arao for review of this manuscript, and Franco DeMayo for his continued support and advice.
ES Cell injection needle | Humagen | MSC-20-0 | |
NSET device | Paratechs | 60010 | |
DMEM | Gibco (life technologies) | 11965-092 | |
FBS | Gibco (life technologies) | 10439-024 | lots should be individually tested for ES Cell compatibility. |
HEPES | Sigma | H0887 | |
b-mercaptoethanol | Sigma | M7522 | |
Micro manipulator | Leica | Micro manipulator | |
micro injector | Eppendorf AG | Cell Tram Air 5176 | |
Microscope | Leica | DM IRB | |
4 well dish | Thermo Scientific | 176740 | |
60 mm dish | Sarstedt | 83.3901 | |
B6(Cg)-Tyr<c-2J>/J | Jackson Labs, Bar Harbor, Maine, USA | 000058 | |
Swiss Webster mice | Taconic, USA | Tac:SW | |
Injection Dish | MatTek Corp. | P50G-0-30-F |