Phenotypic Analysis of Preimplantation Mouse Embryos in Culture
- 1Department of Genetics and Development, Columbia University Medical Center, New York, New York 10032, USA
- 2Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas 77030, USA
- ↵3Correspondence: vep1{at}columbia.edu
Abstract
Preimplantation embryo culture is a valuable approach to investigate a preimplantation lethal phenotype. Standard culture methods have been perfected such that the entire preimplantation period and the process of implantation can be followed in vitro. This protocol provides modifications for the analysis of clutches of embryos from heterozygous matings specifically for the purpose of distinguishing a preimplantation phenotype in homozygous mutants.
INTRODUCTION
In vitro culture of preimplantation mouse embryos is an excellent way of observing events that normally take place in the mother's reproductive tract. Superovulation of female mice can be used to increase the number of embryos obtained per female (Behringer et al. 2018). Protocols for mouse embryo collection, culture media, and culture are fairly standard (see Chapter 4 in Behringer et al. 2014). Preimplantation embryos are quite robust and will survive a range of culture conditions; however, their development is compromised in suboptimal conditions. To recapitulate in vivo development as closely as possible so that a mutant phenotype can be distinguished, rigorous attention must be paid to the details of culture, particularly the culture medium, pH, temperature, and humidity. Although several popular medium formulations have been in use for decades, some of these clearly do not support optimal development and should be avoided. As embryos develop, their culture requirements change. For attachment and further growth of blastocysts in vitro, it is necessary to have serum in the medium and to culture the embryos on tissue culture plastic (as opposed to bacteriological Petri dishes). This protocol provides hints for culturing embryos from the zygote stage to the blastocyst stage for the purpose of assessing mutant phenotypes during the preimplantation period.
MATERIALS
Reagents
Embryo culture medium
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FHM (flushing and handling medium, which contains HEPES and reduced bicarbonate to maintain the correct pH in air) for collecting and handling embryos in air (e.g., MilliporeSigma; also see Chapter 4 in Behringer et al. 2014)
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KSOM + AA for zygote through blastocyst culture (e.g., MilliporeSigma; also see Chapter 4 in Behringer et al. 2014)
Heterozygous male and female mice
Pregnant mice at embryonic day (E) 0.5
Equipment
Dissection instruments
Embryo handling pipette (see Troubleshooting for preparation)
Incubator, humidified, gassed 5% CO2 in air or 5% CO2/5% O2/90% N2, 37°C
Light mineral oil
Microscopes
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Dissecting microscope for embryo handling and evaluation
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Phase or DIC microscope for embryo evaluation
Plastic Petri dishes, 35-mm (or glass watch glasses) for embryo collection
Tissue culture plastic Petri dishes, 35- to 100-mm, for embryo culture
METHOD
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1. Set up heterozygous matings and check plugs daily.
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Either select heterozygous females in estrus and mate with heterozygous males or, alternatively, superovulate immature heterozygous females (Behringer et al. 2018) and set them up with heterozygous males. Check for plugs the following day.
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2. On the day of the plug (E0.5), prepare culture dishes. When comparing embryos of different genotypes, each embryo must be cultured separately for identification purposes. This is easily done by setting up a series of numbered drops of KSOM + AA medium (10–20 µL) in tissue culture Petri dishes and covering them with light mineral oil to keep them separate and minimize evaporation. Equilibrate culture medium drops in a gassed incubator for an hour or so before collecting the zygotes.
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3. Collect zygotes from the oviducts using FHM and remove cumulus cells (see Chapter 4 in Behringer et al. 2014).
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4. Take a sample of tissue from the mother to confirm her genotype.
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5. Place the zygotes individually into equilibrated culture medium drops but identified by litter (this way you can check the genotype of the mother in the interim and discard any litters from mothers who were misgenotyped).
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6. Score each embryo according to the presence or absence of two pronuclei (see Fig. 1B in Overview: Phenotypic Analysis of Preimplantation Lethality in Mice [Papaioannou and Behringer 2023a]) and place in the incubator.
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7. Score the embryos daily with a dissecting microscope or with phase or DIC microscopy, using the classifications listed in the Introduction of Overview: Phenotypic Analysis of Preimplantation Lethality in Mice (Papaioannou and Behringer 2023a). Acquire photographic images for documentation.
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For embryos within the zona pellucida, focus on the zona pellucida until you see a sharp edge, which will be the equatorial plane. For general tips on embryo imaging, see Photodocumentation of Embryos in Overview: Phenotypic Analysis of Periimplantation to Mid-Gestation Lethality (Papaioannou and Behringer 2023b).
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8. At the end of 4–5 d of culture (or less, if an abnormal mutant phenotype is evident earlier), genotype each embryo by polymerase chain reaction (PCR) and correlate genotype with phenotype to confirm the mutant status of abnormal embryos.
As always, if something technical prevents you from genotyping the embryos, you will need to know the normal variation in a control backcross to make a valid statistical comparison (see Protocol: Establishing Control Frequency of Embryonic and Fetal Loss in a Mutant Mouse Colony [Papaioannou and Behringer 2023c]).
TROUBLESHOOTING
Handling embryos is best done with a mouth-controlled Pasteur pipette that has been pulled out over a flame and broken off at an internal diameter of ∼120–150 µm (or just large enough that an embryo fits inside the pipette easily without distortion). Before picking up the embryos, fill the pipette with medium up to the first widening of the pipette bore. You can pick up a large number of embryos at a time in a small volume, but keep them near the tip of the pipette so that they are not lost on the air/medium interface. Monitor all embryo manipulations under a dissecting microscope. Once the embryos are in their individual culture drops, they can be monitored and scored over a period of days. During monitoring, minimize the time out of the incubator to avoid extreme temperature and pH fluctuations.
Make sure the culture drops are completely covered by the mineral oil. If the drop is at the air/oil interface, it will quickly evaporate. Keep the volume of the culture drops low to prevent bubbles of medium from lifting off into the oil when the dish is moved.
DISCUSSION
The starting point for embryo culture can be any time during preimplantation development and can be extended for longer periods to observe the attachment and outgrowth of blastocysts. Isolated inner cell masses (ICMs) can also be cultured to observe the formation of the primitive endoderm. For extended periods of culture and for ICM culture, embryonic stem (ES) cell medium with 10% fetal calf serum should be used as the embryos nutritional requirements change around the time of implantation. If you are culturing from the zygote through blastocyst attachment and outgrowth on the culture dish, the medium should be changed after attachment.
Footnotes
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From the Mouse Phenotypes collection by Virginia E. Papaioannou and Richard R. Behringer.
