The authors would also like to acknowledge Dr. Xiong&#39;s group at Georgia Health Sciences University for manufacturing and sharing the constructs for neogenin cDNA and shRNA vectors. This study was supported by grants from the Basic Science Research Program (2013R1A1A4A01012572) funded by the Korean Research Foundation and by a research grant from Sahmyook University.

NOTE: All procedures for animal breeding and cares were conducted according to the IACUC&#160;regulations of Sahmyook University, Seoul, S. Korea.
1. Superovulation, Breeding and Oviduct Isolation
<ol>
	<li>Maintain inbred C57BL/6 mice under the following conditions: 22 &#177; 3 &#176;C, ~60% humidity, 12 hr light/dark cycle, and water and food ad libitum.</li>
	<li>Induce superovulation of 3-5 week-old female mice by an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (PMSG) at 0.1 ml/head followed 48 hr later by an intraperitoneal injection of 5 IU human chorionic gonadotropin (hCG) at 0.1 ml/head.</li>
	<li>Immediately following hCG injection, mate superovulation-induced females with sexually mature males of the same strain by keeping them in the same cage overnight.</li>
	<li>Next morning, confirm successful mating by the presence of a mating plug or vaginal plug on the female genital tract.</li>
	<li>Sacrifice pregnant female mice by CO2 intoxication followed by cervical dislocation 18-20 hr after hCG injection.</li>
	<li>Open the abdominal cavity by cutting the skin and then the peritoneal layer with fine scissors.</li>
	<li>Pick up the uterine horns with fine forceps and pull away the uterus, oviduct, ovary, and fat pad gently from the body cavity.</li>
	<li>Cut the area between the oviduct and the ovary with fine scissors. Reposition the forceps and cut the uterus near the oviduct, leaving at least 1 cm of the upper part of the uterus attached.</li>
	<li>Transfer the oviductal ampulla to a 35 mm Petri dish containing M16 medium at room temperature. Pool the oviducts from several mice in the same dish.</li>
	<li>Place the dishes under a stereomicroscope for retrieval of embryos.</li>
</ol>
2. Retrieval and Culture of 2-Pronuclear (2-PN) Embryos
<ol>
	<li>Cut the end of a 30 or 32 G hypodermic needle, grind it into a blunt tip, and use it as a flushing needle.</li>
	<li>Use fine forceps to slide the end of the oviduct onto the flushing needle. Gently press the tip of the flushing needle against the bottom of the dish to hold it in place. Flush the oviduct with ~0.1 ml of M16 medium to release the 2-PN embryos into a Petri dish.</li>
	<li>Recover the 2-PN embryos along with surrounding cumulus masses from the flushed M16 medium under the stereomicroscope using a sterile glass pipette and transfer them into a new Petri dish.</li>
	<li>Dissociate cumulus cells from the 2-PN embryos by enzymatic digestion with 1.0 ml of 0.1-0.5% hyaluronidase in Dulbecco&#39;s phosphate-buffered saline (DPBS) at 37 &#176;C for 5-10 min.</li>
	<li>Denude embryos by gently pipetting with a glass pipette and physical removal of cumulus cells.</li>
	<li>Wash denuded embryos three times with 30-50 ml of fresh phosphate-buffered saline (PBS) per embryo.</li>
	<li>Incubate embryos in a 35 mm plastic Petri dish in M16 media supplemented with 10 mg/ml bovine serum albumin (BSA, Fraction V) at 37 &#176;C in 5% CO2 in a humidified incubator until the microinjection is made, which is usually less than 4 hr later.</li>
</ol>
3. Embryo Reverse Transcription (RT) and Polymerase Chain Reaction (PCR)
<ol>
	<li>Collect at least 10 embryos from each stage up to the blastocyst stage and pool them in 4.0 &#956;l of 5x reverse transcriptase pre-mixture solution containing reverse transcriptase, RNase inhibitor, oligo dT primer, random 6mers, dNTP mixture, and the reaction buffer in a PCR tube.</li>
	<li>Add RNase-free distilled H2O to a total volume of 20 &#956;l and sonicate the pooled embryos for 30 sec at 200 W. Initiate the RT reaction immediately at 42 &#176;C for 1 hr followed by 5 min incubation at 95 &#176;C.</li>
	<li>For each 5.0 &#956;l of cDNA solution generated, conduct normal PCR amplification with primers for neogenin, Cdx2, Sox2, Tead4, Nanog, Oct3/4, or &#946;-actin (primer sequences given in Table 1. PCR consists of 40 cycles of 15 sec incubation at 95 &#176;C, 30 sec at annealing temperature (shown in Table 1), and 30 sec at 72 &#176;C.</li>
	<li>Separate PCR products by electrophoresis on a 2% agarose gel and visualize the gel under UV light after staining with ethidium bromide.</li>
	<li>Normalize target gene expression against &#946;-actin levels.</li>
</ol>
4. Immunocytochemistry of Embryos
<ol>
	<li>Fix embryos from each stage with 4% paraformaldehyde in PBS for 30 min at room temperature followed by a brief washing 3 times with PBS containing 0.01% BSA.</li>
	<li>Permeabilize Cell membranes by incubating 10 embryos in 1.0 ml of PBS containing 0.1% Tween 20 and 0.1% TritonX-100 at room temperature for 10 min.</li>
	<li>To block nonspecific binding incubate the permeabilized embryos in 1.0 ml of PBS supplemented with 10% normal goat serum for 30 min at room temperature.</li>
	<li>Incubate the blocked embryos with rabbit anti-neogenin antibodies at 1:100 dilution in PBS containing 0.1% BSA overnight at 4 &#176;C.</li>
	<li>After three washes for 30 sec each in drops of PBS, incubate embryos with either Alexa 488-conjugated goat anti-rabbit IgG or Alexa 568-conjugated goat anti-rabbit IgG at 1:500 dilution in PBS/BSA solution overnight at 4 &#176;C.</li>
	<li>To visualize actin filaments, incubate embryos in 1 &#181;g/ml phalloidin in PBS for 1 hr at room temperature.</li>
	<li>Stain the nuclei by adding 5 ml PBS containing 1 &#181;g/ml DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride) for 5 min at room temperature.</li>
	<li>Wash embryos three times briefly with PBS.</li>
	<li>Transfer embryos to a glass slide and cover them with a drop of mineral oil.</li>
	<li>Take images at 1,000X magnification with a confocal microscope with an excitation of the appropriate wave length.</li>
</ol>
5. Preparation of Neogenin-RFP and Neogenin-siRNA GFP Plasmids
<ol>
	<li>Subclone cDNA encoding mouse neogenin into a Red Fluorescent Protein (RFP)-containing vector, resulting in neogenin-flag-RFP.</li>
	<li>Subclone small hairpin RNA targeting neogenin into an Emerald Green Fluorescent Protein (EmGFP)-containing vector, resulting in pcDNA-EmGFP-miR-neogenin (shown in Figure 1).</li>
	<li>Amplify both neogenin-flag-RFP and pcDNA-EmGFP-miR-neogenin plasmids and purify them with conventional methods.</li>
	<li>Adjust the final concentrations of plasmids to ~1.0 &#181;g/ml in sterile Tris-EDTA (TE) buffer.</li>
</ol>
6. Microinjection of Plasmids into 2-PN Embryos
<ol>
	<li>Wash the denuded 2-PN embryos once, briefly, with fresh M16 media.</li>
	<li>Centrifuge the 2-PN embryos for 10 min at 1,000 x g in a table-top centrifuge to allow for observation of the pronuclei.</li>
	<li>Incubate the embryos in a micro drop of 20-30 &#181;l of M16 media per embryo under mineral oil at 37 &#176;C in 5% CO2 for an additional 1-2 hr.</li>
	<li>Manufacture injection pipettes and holding pipettes by pulling borosilicate-glass capillary tubing (O.D = 1.2 mm; I.D = 0.94 mm) with a mechanical puller.</li>
	<li>Fabricate injection pipettes to have blunt tips and polished openings of &#60;500 &#181;m tip length and 1-2 &#181;m tip inner diameter using a microgrinder and a microforger.</li>
	<li>Fabricate holding pipettes such that they have blunt tips and polished openings with an inner diameter of 20 &#181;m and an outer diameter of 100 &#181;m.</li>
	<li>Load injection pipettes with a sufficient amount (&#62;200 nl) of plasmid solution at 1.0 &#181;g/&#181;l.</li>
	<li>Microinject&#160; ~10 pl of plasmid solution for each 2-PN embryo into one of the pronuclei using a microinjector attached to a motorized micromanipulator; during this process, keep embryos in position by applying negative pressure with a holding pipette.</li>
	<li>After microinjection, wash the 2-PN embryos 3 times with fresh M16 media for less than 1 min each time.</li>
	<li>Culture the 2-PN embryos in a drop of fresh M16 media on a plastic culture dish covered by a mineral oil drop to prevent media evaporation.</li>
	<li>Observe the embryos at 24 hr intervals under a differential interference contrast (DIC) inverted microscope at 200X magnification.</li>
</ol>

<ol>
	<li>Yamanaka, Y., Ralston, A., Stephenson, R. O., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+molecular+regulation+of+the+mouse+blastocyst.">Cell and molecular regulation of the mouse blastocyst.</a> Dev. Dyn. 235 (9), 2301-2314 (2006).</li><li>Sasaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+trophectoderm+fate+specification+in+preimplantation+mouse+development.">Mechanisms of trophectoderm fate specification in preimplantation mouse development.</a> Dev. Growth Differ. 52 (3), 263-273 (2010).</li><li>Nishioka, N., Yamamoto, S., Kiyonari, H., Sato, H., Sawada, A., Ota, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tead4+is+required+for+specification+of+trophectoderm+in+pre-implantation+mouse.">Tead4 is required for specification of trophectoderm in pre-implantation mouse.</a> Mech. Dev. 125 (3-4), 270-283 (2008).</li><li>Ovitt, C. E., Sch&#246;ler, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+Oct-4+in+the+early+mouse+embryo.">The molecular biology of Oct-4 in the early mouse embryo.</a> Mol. Hum. Reprod. 4 (11), 1021-1031 (1998).</li><li>Stein, P., Schindler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Oocyte+Microinjection,+Maturation+and+Ploidy+Assessment.">Mouse Oocyte Microinjection, Maturation and Ploidy Assessment.</a> J. Vis. Exp. (53), e2851 (2011).</li><li>Shin, M. R., Cui, X. S., Jun, J. H., Jeong, Y. J., Kim, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+mouse+blastocyst+genes+that+are+down+regulated+by+double-stranded+RNA-mediated+knockdown+of+Oct-4+expression.">Identification of mouse blastocyst genes that are down regulated by double-stranded RNA-mediated knockdown of Oct-4 expression.</a> Mol. Reprod. Dev. 70 (4), 390-396 (2005).</li><li>Khang, I., Sonn, S., Park, J. -. H., Rhee, K., Park, D., Kim, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+epithin+in+mouse+preimplantation+development:+Its+role+in+compaction.">Expression of epithin in mouse preimplantation development: Its role in compaction.</a> Dev. Biol. 281, 134-144 (2005).</li><li>Prieto, D., Aparicio, G., Machado, M., Zolessi, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+DNA-Specific+Stain+Methyl+Green+in+the+Fluorescent+Labeling+of+Embryos.">Application of the DNA-Specific Stain Methyl Green in the Fluorescent Labeling of Embryos.</a> J. Vis. Exp. (99), e52769 (2015).</li><li>Lee, J. H., Choi, S. S., Kim, H. W., Xiong, W. C., Min, C. K., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neogenin+as+a+receptor+for+early+cell+fate+determination+in+preimplantation+mouse+embryos.">Neogenin as a receptor for early cell fate determination in preimplantation mouse embryos.</a> PLoS One. 9 (7), e101989 (2014).</li><li>Gordon, J. W., Scangos, G. A., Plotkin, D. J., Barbosa, J. A., Ruddle, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+transformation+of+mouse+embryos+by+microinjection+of+purified+DNA.">Genetic transformation of mouse embryos by microinjection of purified DNA.</a> Proc. Natl. Acad. Sci. USA. 77 (12), 7380-7384 (1980).</li><li>Brinster, R. L., Chen, H. Y., Trumbauer, M. E., Yagle, M. K., Palmiter, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+the+efficiency+of+introducing+foreign+DNA+into+mice+by+microinjecting+eggs.">Factors affecting the efficiency of introducing foreign DNA into mice by microinjecting eggs.</a> Proc. Natl. Acad. Sci. USA. 82, 4438-4442 (1985).</li></ol>

The authors have no competing financial interests to disclose.

In the present protocol, we demonstrate novel methods of microinjection of genetic materials, either cDNA or shRNA, into the 2-PN mouse embryos to explore the role of neogenin in early cell differentiation during mouse embryogenesis. Genetic modification of preimplantation embryos is a powerful technique in uncovering key information about underlying molecular mechanisms for, for example, the first cell lineage determination. Genetic modification is one of the most commonly used methods for ascertaining gene function. Wi...

Preimplantation embryonic development can be divided into several distinct stages, from the one-cell stage to the morula and the blastocyst. Much evidence suggests that polarity and positional cues play a role in early cell fate determination into the trophectoderm (TE) and the inner cell mass (ICM). However, the nature of the cues, how they are transduced into cellular signals, and the physiological contexts in which such signals are capable of initiating cell lineage differentiation are not known. These differentiated cells then undergo specialization, beginning to take on characteristic structures and functions needed for embryo proper formation and growth of the placenta1-3.
Pre-implantation embryos are particularly amenable to manipulation by direct injection of modified genetic materials like siRNA or target cDNA and thus can be utilized to study specific target molecules. There is a growing understanding that genetic analysis of vertebrate embryos is critical to advancing our understanding of embryonic development4. Precise introduction of a wild-type gene or a mutant form into an embryo in a timely context to accomplish gain- or loss-of-function of the gene has greatly facilitated the study of developmentally regulated genes. Loss- and gain-of-function via microinjection of genetic materials into individual early non-mammalian and mammalian embryos is relatively simple because of their large size and great receptivity5. Microinjection is typically performed using non-viral vectors. Particular advantage has been taken of the ease of using non-viral vectors over viral vectors and transgenes. A rapid loss- or gain-of-function expression system in mouse embryos has been developed that allows us to dissect and understand gene functions in vertebrate embryology6,7.
Fluorescent labeling of embryos would greatly facilitate the selection of microinjected or genetically manipulated embryos and at the same time grant a means to indirectly quantify the expression level of microinjected siRNA or cDNA based on fluorescent intensity8. To accomplish this labeling, fluorescent protein cDNA, GFP or RFP, are co-injected as either fusion constructs or separately. The fluorescence intensity of GFP or RFP reveals the extent of expression of siRNA or foreign DNA to ensure that loss- or gain-of-function has been accomplished.
Here, we present a micromanipulation protocol for loss- and gain-of- function technique that allowed us to identify neogenin as a receptor that relays extracellular cues important in early cell differentiation in preimplantation mouse embryos.

<table><tbody><tr><td>M16 medium,</td><td>Gibco BRL (Grand Island, NY)</td><td>M7292 LOT# 11A832</td><td></td></tr><tr><td>Goat serum,&amp;nbsp;</td><td>Dako (Glostrup, Denmark)</td><td>X0907</td><td></td></tr><tr><td>PMSG</td><td>Folligon, Intervet, Holland</td><td>G4877-1000IU LOT#SLBD0719V</td><td></td></tr><tr><td>Mineral oil</td><td>Sigma Aldrich</td><td>CG5-1VL LOT# SLBC6783V</td><td></td></tr><tr><td>human chorionic gonadotropin and pregnant mare serum gonadotropin&amp;nbsp;</td><td>(Intervet, Holland)</td><td>(invitrogen , 15596-026)</td><td></td></tr><tr><td>SuperScript&amp;reg; III Reverse Transcriptase</td><td>lifetechnologies</td><td>18080-044</td><td></td></tr><tr><td>PCR pre mixture</td><td>&amp;nbsp;(Bioneer, Daejon, S. Korea)</td><td>GenDEPOT , A0224-050</td><td></td></tr><tr><td>Agarose (molecular grade)</td><td>BioRad</td><td>161-3101</td><td></td></tr><tr><td>Paraformaldehyde Solution, 4% in PBS</td><td>Affymetrix USA</td><td>19943</td><td></td></tr><tr><td>polyclonal rabbit anti-neogenin antibody</td><td>Santa Cruz Biotechnology, US</td><td>SC-15337</td><td></td></tr><tr><td>Alexa fluor 488 labeled anti-rabbit antibody</td><td>Molecular Probes (Invitrogen, USA)</td><td>A-11094</td><td></td></tr><tr><td>Alexa fluor 555 labeled anti-rabbit antibody</td><td>Molecular Probes (Invitrogen, USA)</td><td>A-21428</td><td></td></tr><tr><td>Alexa Fluor&amp;reg; 555 Phalloidin</td><td>Molecular Probes (Invitrogen, USA)</td><td>A34055</td><td></td></tr><tr><td>DAPI (4&#039;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td><td>Invitrogen, USA</td><td>D1306</td><td></td></tr><tr><td>Recombinant Human RGM-C/Hemojuvelin</td><td>R&amp;amp;D systems</td><td>3720-RG</td><td></td></tr><tr><td>all plasmid constructs&amp;nbsp;</td><td>Prof. Wen Cheng Xiong at Georgia Health Sciences University (Agusta, GA).</td><td></td><td>for the present studies were kindly provided by&amp;nbsp;</td></tr><tr><td>Neon&amp;reg;&amp;nbsp; Transfection System</td><td>lifetech</td><td>MPK10096</td><td></td></tr><tr><td>Stereo Microscrope</td><td>Nikon</td><td>SMZ1000</td><td></td></tr><tr><td>Micromainpulation system with Nikon</td><td>Nikon</td><td>Narishige ONM-1</td><td></td></tr><tr><td>Micro Injector</td><td>Nikon Narishige</td><td>GASTIGHT #1750</td><td></td></tr><tr><td>Holder</td><td>Nikon Narishige</td><td>Narishige IM 16</td><td></td></tr><tr><td>Microfuge</td><td>Nikon Narishige</td><td>Narishige MF-900</td><td></td></tr><tr><td>grinder</td><td>Narishige</td><td>EG400</td><td></td></tr><tr><td>Puller</td><td>Sutter Instrumnet company</td><td>P-97</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Thermo Scientific Forma</td><td>EW-39320-16</td><td></td></tr><tr><td>Veriti&amp;reg; 384-Well Thermal Cycler&amp;nbsp;</td><td>lifetechnologies</td><td>4388444</td><td></td></tr></tbody></table>

loss- and gain-of-function approach to investigate early cell fate determinants in preimplantation mouse embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

We discovered that neogenin is transiently expressed during the early developmental stages of preimplantation mouse embryos, appearing as early as at the 2-cell stage and lasting until the early morula but becoming deficient at the late morula and blastocyst stages (Figure 2A). In addition, the spatial distribution of neogenin was restricted mainly to outside cells. The results of RT-PCR analysis were consistent with the early and transient nature of neogenin expression, ...

Watch this Scientific Journal Video about Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos at JoVE.com

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

loss-gain-function-approach-to-investigate-early-cell-fate

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

8.7K Views.  Dankook University. The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Video: Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Methods for Precisely Localized Transfer of Cells or DNA into Early Postimplantation Mouse Embryos

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, cell culture has been widely used in developmental biology studies. However, it is important to determine whether in vitro cultured cells truly represent in vivo cell types. Grafting cells into embryos, followed by an assessment of their contribution during development is a useful method to determine the potential of in vitro cultured cells. In this study, we describe a method for grafting cells into a defined site of early postimplantation mouse embryos, followed by ex vivo culture. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing precise localization and adjustment of the number of cells receiving exogenous DNA with both high transfection efficiency and low cell death. These techniques, which do not require any specialized equipment, render experimental manipulations of the gastrulation and early organogenesis-stage mouse embryo possible, allowing analysis of commitment in cultured cell subpopulations and the effect of genetic manipulations in situ on cell differentiation.

We demonstrate a method for grafting cultured cells into defined sites of early mouse embryos to determine their in vivo potential. We also introduce an optimized electroporation method that uses glass capillaries of known diameter, allowing the precise delivery of exogenous DNA into a few cells in the embryos.

Manipulation and culture of early mouse embryos is a powerful yet largely under-utilized technology enhancing the value of this model system. Conversely, ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Genetics

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal which tissues descend from each cell of the embryo. Although fate maps are very useful for identifying the precursors of an organ and for elucidating the developmental path by which the descendant cells populate that organ in the normal embryo, they do not illustrate the full developmental potential of a precursor cell or identify the mechanisms by which its fate is determined. To test for cell fate commitment, one compares a cell's normal repertoire of descendants in the intact embryo (the fate map) with those expressed after an experimental manipulation. Is the cell's fate fixed (committed) regardless of the surrounding cellular environment, or is it influenced by external factors provided by its neighbors? Using the comprehensive fate maps of the Xenopus embryo, we describe how to identify, isolate and culture single cleavage stage precursors, called blastomeres. This approach allows one to assess whether these early cells are committed to the fate they acquire in their normal environment in the intact embryo, require interactions with their neighboring cells, or can be influenced to express alternate fates if exposed to other types of signals.

The fate of an individual embryonic cell can be influenced by inherited molecules and/or by signals from neighboring cells. Utilizing fate maps of the cleavage stage Xenopus embryo, single blastomeres can be identified for culture in isolation to assess the contributions of inherited molecules versus cell-cell interactions.

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Biology

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

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Mouse In Vivo Placental Targeted CRISPR Manipulation

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Mouse Genome Engineering Using Designer Nucleases