Fertilizing and modifying mouse embryos

[Calvin P]

Hello everyone I'm currently a student so apologies in advance if I'm incorrect or describe what I'm trying to do poorly, but essentially I'm kind of confused on how extracted mice embryo are fertilized and genetically modified during in vitro fertilization.  To be a little more specific consider the following situation and again if I'm wrong about anything below please correct me:

1. A pre-pregant female mouse is euthanized and then dissected to retrieve it's embryo.

2. A mammalian plasmid vector such as pcDNA3 with a cationic liposome coating is added to the embryo so the embryonic cell can uptake the plasmid DNA.  One of the things I'm confused about is how the embryo is stored and how the plasmid vector is introduced to that solution.

3. The embryo is fertilized. Not sure how this occurs.

4. Embryo is implanted into a female mouse using an nset device.

 

Any guidance would be greatly appreciated, thank you in advance for your time.

[Dagl1]

Some points, what is an embryo? Embryonic development happens after fertilization. I presume you mean egg cell?

Egg cells can be frozen, and are just in solution when a plasmid is added. Some transfection reagent can then be added (Fugene) together with the plasmid to transfect the cells: 

Eggs were exhaustively washed in M2 and then transferred to 50 µl microdrops of M2 containing the mixture of DNA and liposomes, under mineral oil. The plasmid used for the transfection was a commercial plasmid, pGeneGrip (GTS, CA) that encodes the green fluorescent protein (GFP) under the control of hCMV IE promoter/enhancer and is rhodamine labeled. For each experiment 125 ng of DNA in combination with 1 µl of a commercial liposome mixture (Fugene, BoehringerManheim) were resuspended in 100 µl of M2. Transfection was allowed to proceed for 3 hr at 37°C in an atmosphere of 5% CO2. Controls using Fugene only or the plasmid only were carried out.

Sperm is collected, diluted into medium, and then added to the cells to fertilize them.

Sperm were collected from the caudae epididymides of a vigorous stud male. Both caudae were punctured in 200 µl of pre-warmed capacitation medium (M16 medium supplemented with 40 mg/ml of bovine seroalbumin [BSA] and antibiotics) Sperm were diluted in capacitation medium to give a final concentration of 1–2 3 106 cells/ml. Capacitation took place for 3 hr at 37°C in an atmosphere of 5% CO2. At the end of the incubation, 100 µl drops from the sperm suspension were placed under mineral oil and the transfected eggs were transferred to these drops and left overnight.Twenty-two to twentyfour hours later the inseminated eggs were examined for signs of nuclear decondensation.... etc.

 

Does that answer your questions? (note that I just found the first article that described this, nowadays there may be many different methods, but they probably follow the same base steps).

From https://www.ncbi.nlm.nih.gov/pubmed/10862002  Carballada, Rosa, Tedla Degefa, and Pedro Esponda. "Transfection of mouse eggs and embryos using DNA combined to cationic liposomes." Molecular Reproduction and Development: Incorporating Gamete Research 56.3 (2000): 360-365.

 

[Calvin P]

@Dagl1 Thank you very much for the insights they we're all incredibly helpful, however I still have a few more questions.

1. What's M2 and how do you "wash" egg cells in them?

2. I'm a bit confused on the mixture that the egg cells are stored in after I'm wondering if the following ratios are correct and if I'm missing anything
    100 µl  - M2 Solution
    125ng  - DNA plasmids (ie: pGeneGrip, pcDNA3)
    1x         - Egg cell
    1 µl       - Liposomes (Fugene, BoehringerManheim)
    ????      - mineral oil

Edited April 25, 2020 by Calvin P

[Dagl1]

Washing probably is just putting the egg in the solution, then remove the solution, put another dose of solution, rinse and repeat x amount of times.

https://www.sigmaaldrich.com/catalog/product/sigma/m7167?lang=en&region=NL&gclid=CjwKCAjwv4_1BRAhEiwAtMDLssbOtt671TSOSgmEJMC_4LWNIwG0EaaufWtwvujan2GZvopDMC6wRhoC3oUQAvD_BwE

 mineral oil: https://www.fertstert.org/article/S0015-0282(18)30177-8/fulltext

Regarding the ratios, you may want to search for some methods in newer papers that use this technique, to find if newer methods use different ratios.

[Calvin P]

Ah makes sense, ty.