Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

Summary

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.

Abstract

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.

Introduction

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/Cas9^1,2 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 cells^3,4. Hig....

Protocol

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

1. Breed donor mice, B6(Cg)-Tyr<c-2J>/J female mice at 8 - 10 weeks of age naturally to B6(Cg)-Tyr<c-2J>/J male mice between 8 and 26 weeks of age. Check plugs visually the following morning (E0.5).
2. Breed recipient mice, Swiss Webster....

Results

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.......

Discussion

It has long been thought that any disturbance of the ICM could lead to mortality, in fact, current blastocyst microinjection protocols still warn of this^12,13. 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, shoul.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
β-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