Name: Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm (Video) | JoVE Journal
Uploaded: 2020-11-19T15:00:00.000+00:00
Duration: 8 min 8 s
Description: Learn to use parthenogenetic haploid embryonic stem cells as a sperm substitute for creating semi-cloned mouse embryos. This method enables genetic manipulation to study genomic imprinting in vivo.

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We thank Dr. Giulio Di Minin for derivation of phaESC lines and Dr. Remo Freimann for flow cytometry operation. We also acknowledge Ms. Mich&#232;le Schaffner and Mr. Thomas M. Hennek for technical support with embryo transfer. This work was supported by the Swiss National Science Foundation (grant 31003A_152814/1).

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</ol>

The authors have nothing to disclose.

Cloning of mammals by somatic cell nuclear transfer (SCNT) has been pioneered in the 1990s19,20,21. These developments followed cloning studies conducted 30 years earlier in amphibians22. The considerable delay reflects the difficulty of embryology and genomic imprinting in mammals. The development of mammalian SCNT is the basis for the application of haESC for substituting sperm, which is detailed in this protocol.
Cell cycle synchronization is an important factor for the success of SCNT23. This is also the case for injection of haESC in this protocol. Introducing a donor genome into a recipient requires the cell cycle phases to be matched to avoid chromosomal breakage or aneuploidies that would abrogate embryo development. Semi-cloning has the additional complexity that two genomes and a cytoplast need to be compatible. A previous report has demonstrated that injection of M-phase-arrested androgenetic haESCs yielded better developmental rates of semi-cloned embryos than injection of G1-phase haESCs into oocytes7. This report suggests M-phase as a suitable synchronization point for semi-cloning. Accordingly, phaESCs were mitotically arrested in metaphase with demecolcine and injected into the ooplasm of MII oocytes, which were naturally arrested in metaphase II of meiosis. Importantly, M-phase arrest can be achieved in mouse ESCs with high efficiency, providing excellent synchronization between donor and recipient cell cycles.
During mitosis, the nuclear membrane breaks down and a spindle forms to which the replicated chromosomes attach. After injection of M-phase DKO-phaESCs, sister chromatids are segregated into a pseudo polar body and the zygote7 (Figure 3E). Consequently, a single set of chromosomes from a DKO-phaESC contributes to the semi-cloned embryo. It is critical that the sister chromatids of DKO-phaESC chromosomes can be correctly segregated after injection. The plasma membrane of an intact DKO-phaESC prevents segregation into a pseudo polar body. We did indeed observe rare cases where the plasma membrane of DKO-phaESCs was not ruptured, and embryos exhibited intact DKO-phaESCs in the ooplasm after injection (Figure 3D). Therefore, care must be taken to remove the plasma membrane of DKO-phaESCs by pipetting. During injection, it is equally important to avoid the disruption of the meiotic spindle of the oocyte, which could lead to chromosome segregation defects and induce aneuploidy as well.
In mammals, genomic imprinting limits the application of phaESCs as sperm replacement. Parthenogenetic haESCs possess a maternal configuration of genomic imprints, whereas sperm possess a paternal configuration. Therefore, generation of full-term pups has not occurred after the injection of wildtype phaESCs as sperm replacement. To overcome this limitation, deletions of the IG- and H19-DMRs are engineered in phaESCs. Modification of imprinted expression at the maternal Igf2-H19 and Gtl2-Dlk1 loci is sufficient to change the configuration of genomic imprints for allowing the generation of semi-cloned mice with a frequency of over 5.1%, based on transferred 2-cell embryos. These observations suggest that targeting two imprinted genes switches the genome of phaESCs into a functional paternal configuration that can replace sperm in mice. Nevertheless, a permanent genetic modification of the phaESCs is required for this strategy. As an alternative strategy, androgenetic haESC can be considered. Androgenetic haESCs are derived from the sperm genome and possess paternal imprints. There have been reports that wildtype androgenetic haESCs contributed as sperm replacement to generate full-term pups at a frequency between 1.3% and 1.9% of transferred embryos4,7,24. Full-term pups have also been obtained by injecting androgenetic haESCs with deletions of the IG- and H19-DMRs at a frequency of 20.2% of transferred 2-cell embryos24. The increased efficiency of semi-cloning using modified androgenetic haESCs is likely because imprints can become unstable in culture. Imprinting defects are corrected by the genetic deletions of critical DMRs.
Considering the difficulty in introducing genetic modifications directly into the oocyte or sperm genomes, haESCs are a valuable tool for manipulating the parental genomes separately. Using haESCs as a substitute of sperm provides a remarkable advantage for genome editing in the mouse germline. A recent study has combined CRISPR/Cas9-based genome editing with the application of haESCs for the characterization of imprinting regions that are critical for embryonic development12. This study analyzed the role of the Rasgrf1-DMR in combination with the H19-and IG-DMRs in the development of bimaternal mice, and the function of 7 different DMRs in the development of bipaternal mice. The method for substituting haESCs for sperm formed the basis for genetic screening approaches for identifying key amino acids within the DND1 protein in primordial germ cell development and for identifying genes in bone development24,25,26. Studies on genomic imprinting and genetic screening to identify key factors in embryonic development are considerable&#160;approaches for the application of haESCs as gametic genomes.

In mammals, gametes are the only cells that transmit genetic information to the next generation. Fusion of an oocyte and a spermatozoon forms a diploid zygote that develops into an adult animal. Mutations in gametic genomes are thereby inherited by the offspring and drive genetic variation in species1. Introduction of mutations in the germline has been applied to produce genetically modified animals for diverse biological studies including the characterization of gene function and disease modelling. Both oocytes and spermatozoa are terminally differentiated and highly specialized cells that have ceased proliferation. Therefore, direct modification of gametes is technically difficult, and specialized approaches have been developed. Genetic modifications can be introduced into the mouse germline by the injection of genetically modified ESCs into blastocysts, where they integrate into the developing embryo and colonize the germline. Additionally, genetic modification of zygotes using genome editing approaches, including the CRISPR/Cas9 system, has become widely adopted2.
Recently, an outstanding approach has been reported, which applies haESCs as a substitute for a gametic genome3,4,5,6,7,8. HaESCs are stem cell lines derived from the inner cell mass of parthenogenetic or androgenetic haploid blastocysts and possess a single set of chromosomes4,7,9,10. It has been demonstrated that both parthenogenetic and androgenetic haESCs can contribute to the genome of semi-cloned mice after intracytoplasmic injection into oocytes. In contrast to other approaches, the genomes of haESCs can be directly modified in culture owing to their self-renewal capacity. Introducing genetic modifications into the germline by replacing sperm with haESCs is an important method for biological studies. It provides for a possibility to separately culture and manipulate the maternal or paternal genome, which are derived from parthenogenetic or androgenetic haESCs, respectively. HaESCs can then be used as gametic genome replacement, which is especially advantageous for studies of genomic imprinting, allele-specific expression, and parental specific processes.
In mice, both maternal and paternal genomic information is required for normal embryo development11. Therefore, full-term pups could not be obtained when wild-type parthenogenetic haESCs (phaESCs) were injected to replace the sperm genome5,8. To overcome the developmental block, genomic imprinting of the maternal genome of parthenogenetic haESCs needs to be corrected to a paternal configuration. This can be achieved by manipulation of the differentially methylated regions (DMRs). To date, targeted deletions of the H19-DMR, Gtl2-Dlk1 IG-DMR, and Rasgrf1-DMR have been studied to repress maternally expressed genes in phaESCs3,5,8,12. These studies demonstrated that deletions of both the H19-DMR and the IG-DMR are sufficient to convert a maternal into a paternal imprint configuration that can substitute for sperm chromosomes. Intracytoplasmic injection of phaESCs that carry the two DMR deletions into oocytes yielded semi-cloned pups with a frequency between 5.1% and 15.5% of transferred 2-cell embryos.
This protocol is based on the application of phaESCs with deletions of both H19-DMR and IG-DMR, which we term double-knockout phaESCs (DKO-phaESCs). We provide detailed instructions for the modification of genomic imprinting in phaESCs to establish DKO-phaESC lines, for the injection of DKO-phaESCs into oocytes as a substitute for a sperm genome, for culture of semi-cloned embryos to blastocysts, and for obtaining semi-cloned mice. This protocol is a reference for researchers who require precise and direct manipulation of the paternal genome and generation of semi-cloned embryos and mice.

<table><tbody><tr><td>1.5 mLTube</td><td>Eppendorf</td><td>0030 125.150</td><td>haESC culture</td></tr><tr><td>15 ml Tube</td><td>Greiner Bio-One</td><td>188271</td><td>haESC culture</td></tr><tr><td>12-well plate</td><td>Thermo Scientific</td><td>150628</td><td>haESC culture</td></tr><tr><td>24-well plate</td><td>Thermo Scientific</td><td>142475</td><td>haESC culture</td></tr><tr><td>6-well plate</td><td>Thermo Scientific</td><td>140675</td><td>haESC culture</td></tr><tr><td>96-well plate</td><td>Thermo Scientific</td><td>167008</td><td>haESC culture</td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Merck</td><td>M6250</td><td>haESC culture</td></tr><tr><td>BSA fraction</td><td>Gibco</td><td>15260-037</td><td>haESC culture</td></tr><tr><td>CHIR 99021</td><td>Axon</td><td>1386</td><td>haESC culture</td></tr><tr><td>Demecolcine solution</td><td>Merck</td><td>D1925</td><td>haESC culture</td></tr><tr><td>DMEM</td><td>Gibco</td><td>41965-039</td><td>haESC culture</td></tr><tr><td>DMEM / F-12</td><td>Gibco</td><td>11320-033</td><td>haESC culture</td></tr><tr><td>DR4 mouse</td><td>The Jackson Laboratory</td><td>3208</td><td>haESC culture</td></tr><tr><td>EDTA</td><td>Merck</td><td>E5134</td><td>haESC culture</td></tr><tr><td>FBS</td><td>biowest</td><td>S1810-500</td><td>haESC culture</td></tr><tr><td>Freezing medium</td><td>amsbio</td><td>11897</td><td>haESC culture</td></tr><tr><td>Gelatin</td><td>Merck</td><td>G1890</td><td>haESC culture</td></tr><tr><td>Hepes solution</td><td>Gibco</td><td>15630-056</td><td>haESC culture</td></tr><tr><td>Irradiated MEF</td><td>Gibco</td><td>A34966</td><td>haESC culture</td></tr><tr><td>LIF (Leukemia inhibitory factor)</td><td>(Homemade)</td><td>-</td><td>haESC culture</td></tr><tr><td>Lipofection reagent</td><td>Invitrogen</td><td>11668019</td><td>haESC culture</td></tr><tr><td>NDiff 227</td><td>Takara</td><td>Y40002</td><td>haESC culture</td></tr><tr><td>PD 0325901</td><td>Axon</td><td>1408</td><td>haESC culture</td></tr><tr><td>Pen Strep</td><td>Gibco</td><td>15140-122</td><td>haESC culture</td></tr><tr><td>piggyBac plasmid carrying a CAG-EGFP transgene</td><td>Addgene</td><td>#40973</td><td>haESC culture</td></tr><tr><td>piggyBac transposase plasmid</td><td>System Biosciences</td><td>PB210PA-1</td><td>haESC culture</td></tr><tr><td>pX330 plasmid</td><td>Addgene</td><td>#42230</td><td>haESC culture</td></tr><tr><td>pX458 plasmid</td><td>Addgene</td><td>#48138</td><td>haESC culture</td></tr><tr><td>Trypsin</td><td>Gibco</td><td>15090-046</td><td>haESC culture</td></tr><tr><td>DNA polymerase</td><td>Thermo Scientific</td><td>F549S</td><td>Genotyping</td></tr><tr><td>dNTP Mix</td><td>Thermo Scientific</td><td>R0192</td><td>Genotyping</td></tr><tr><td>Ethanol</td><td>Merck</td><td>100983</td><td>Genotyping</td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100317</td><td>Genotyping</td></tr><tr><td>Isopropanol</td><td>Merck</td><td>109634</td><td>Genotyping</td></tr><tr><td>Proteinase K</td><td>Axon</td><td>A3830</td><td>Genotyping</td></tr><tr><td>SDS</td><td>Merck</td><td>71729</td><td>Genotyping</td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>106404</td><td>Genotyping</td></tr><tr><td>ThermoMixer</td><td>Epeendorf</td><td>5384000012</td><td>Genotyping</td></tr><tr><td>Tris</td><td>Merck</td><td>T1503</td><td>Genotyping</td></tr><tr><td>5 mL Tube</td><td>Falcon</td><td>352063</td><td>Flow cytometry</td></tr><tr><td>5 mL Tube with cell strainer cap</td><td>Falcon</td><td>352235</td><td>Flow cytometry</td></tr><tr><td>Hoechst 33342</td><td>Invitrogen</td><td>H3570</td><td>Flow cytometry</td></tr><tr><td>MoFlo Astrios EQ</td><td>Beckman Coulter</td><td>B25982</td><td>Flow cytometry</td></tr><tr><td>1 mL syringe</td><td>Codan</td><td>62.1640</td><td>Superovulation</td></tr><tr><td>30-gauge 1/2 inch needle</td><td>Merck</td><td>Z192362-100EA</td><td>Superovulation</td></tr><tr><td>B6D2F1 mouse</td><td>The Jackson Laboratory</td><td>100006</td><td>Superovulation</td></tr><tr><td>hCG</td><td>MSD animal health</td><td>49451</td><td>Superovulation</td></tr><tr><td>PBS</td><td>Gibco</td><td>10010015</td><td>Superovulation</td></tr><tr><td>PMSG</td><td>Avivasysbio</td><td>OPPA1037 5000 IU</td><td>Superovulation</td></tr><tr><td>10-cm dish</td><td>Thermo Scientific</td><td>150350</td><td>Embryo manipulation</td></tr><tr><td>4-well plate</td><td>Thermo Scientific</td><td>179830</td><td>Embryo manipulation</td></tr><tr><td>50 mL tube</td><td>Greiner Bio-One</td><td>227261</td><td>Embryo manipulation</td></tr><tr><td>6 cm dish</td><td>Thermo Scientific</td><td>150288</td><td>Embryo manipulation</td></tr><tr><td>Center-well dish</td><td>Corning</td><td>3260</td><td>Embryo manipulation</td></tr><tr><td>Digital rocker</td><td>Thermo Scientific</td><td>88880020</td><td>Embryo manipulation</td></tr><tr><td>EGTA</td><td>AppliChem</td><td>A0878</td><td>Embryo manipulation</td></tr><tr><td>Hyaluronidase</td><td>Merck</td><td>H4272</td><td>Embryo manipulation</td></tr><tr><td>KSOM</td><td>Merck</td><td>MR-020P-5F</td><td>Embryo manipulation</td></tr><tr><td>M16 medium</td><td>Merck</td><td>M7292</td><td>Embryo manipulation</td></tr><tr><td>M2 medium</td><td>Merck</td><td>M7167</td><td>Embryo manipulation</td></tr><tr><td>Mineral oil</td><td>Merck</td><td>M8410</td><td>Embryo manipulation</td></tr><tr><td>Glass capillary</td><td>Merck</td><td>P1049-1PAK</td><td>Embryo manipulation</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ745</td><td>Embryo manipulation</td></tr><tr><td>Strontium chloride</td><td>Merck</td><td>13909</td><td>Embryo manipulation</td></tr><tr><td>5ml syringe</td><td>Codan</td><td>62.5607</td><td>Microinjection</td></tr><tr><td>Borosilicate glass cappilary</td><td>Science product</td><td>GB100T-8P</td><td>Microinjection</td></tr><tr><td>CellTram 4r Oil</td><td>Eppendorf</td><td>5196000030</td><td>Microinjection</td></tr><tr><td>Fluorinert FC-770</td><td>Merck</td><td>F3556</td><td>Microinjection</td></tr><tr><td>Holding pipette</td><td>BioMedical Instruments</td><td>-</td><td>Microinjection</td></tr><tr><td>Inverted microscope</td><td>Nikon</td><td>Eclipse Ti-U</td><td>Microinjection</td></tr><tr><td>Microforge</td><td>Narishige</td><td>MF-900</td><td>Microinjection</td></tr><tr><td>Microinjection pipette</td><td>BioMedical Instruments</td><td>-</td><td>Microinjection</td></tr><tr><td>Microlaoder tips</td><td>Eppendorf</td><td>5252 956.003</td><td>Microinjection</td></tr><tr><td>Micromanipulaor arms</td><td>Narishige</td><td>NT-88-V3</td><td>Microinjection</td></tr><tr><td>Micropipette puller</td><td>Sutter Instrument</td><td>P-1000</td><td>Microinjection</td></tr><tr><td>PiezoXpert</td><td>Eppendorf</td><td>5194000016</td><td>Microinjection</td></tr><tr><td>PVP (Polyvinylpyrrolidine)</td><td>Merck</td><td>PVP360</td><td>Microinjection</td></tr><tr><td>Syringe filter</td><td>SARSTEDT</td><td>83.1826.001</td><td>Microinjection</td></tr></tbody></table>

application of mouse parthenogenetic haploid embryonic stem cells as a substitute of sperm

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 ...

application-mouse-parthenogenetic-haploid-embryonic-stem-cells-as

Research

JoVE Journal

Developmental Biology

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Isolation and Derivation of Mouse Embryonic Germinal Cells

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Here we demonstrate the newly developed I•Cryo kit for mouse sperm cryopreservation.  Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit.  Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later successfully recovered by IVF.

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generation of Mice Derived from Induced Pluripotent Stem Cells

This article describes a protocol to efficiently derive and culture pluripotent stem cell lines from mouse embryos at the blastocyst stage.

Derivation of Stem Cell Lines from Mouse Preimplantation Embryos

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.

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

Here, we present a modified method for cryopreservation of one-cell embryos as well as a protocol that couples the use of freeze-thawed embryos and electroporation for the efficient generation of genetically modified mice.

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting method.

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

The cervical manipulation method presented can induce pseudopregnancy in mice without the need for breeding females with vasectomized males. The induction of pseudopregnancy is required for the success of non-surgical embryo transfer and non-surgical artificial insemination, both of which are also presented.

Inducing Pseudopregnancy in Female Mice Without the Need for Vasectomized Males Prior to Non-Surgical Embryo Transfer or Artificial Insemination

Here, we introduce a method for performing round spermatid injection (ROSI) in mice, a technique with promising clinical applications and utility for investigating the mechanisms underlying embryonic development.

Mouse Round Spermatid Injection

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

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

Learn to use parthenogenetic haploid embryonic stem cells as a sperm substitute for creating semi-cloned mouse embryos. This method enables genetic manipulation to study genomic imprinting in vivo.

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

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