Name: application of mouse parthenogenetic haploid embryonic stem cells as a substitute of sperm
Uploaded: 2020-11-19T15:00:00.000+00:00
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Description: Watch this Scientific Journal Video about Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm at JoVE.com

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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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

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.

The purpose of this protocol is to apply haESCs as a substitute of sperm to obtain semi-cloned embryos and mice. For this purpose, a DKO-phaESC line carrying the CAG-EGFP transgene was generated and used for intracytoplasmic injection into MII oocytes. To obtain a suitable phaESC line with a paternal imprint configuration, we performed genetic engineering using Cas9 nucleases. A haploid ESC line contains haploid and diploid cells that arise due to an inherent tendency of haploid ESCs for diploidization10. A haploid chromosome set is a prerequisite for successful replacement of the sperm genome. DNA content analysis by flow cytometry shows the distribution of haploid and diploid cells at G0/G1-, S-, and G2/M-phases (Figure 2A).
To establish the DKO-phaESC lines, wildtype phaESC lines were transfected with piggyBac transposon constructs for stable transgenic EGFP expression and with CRISPR/Cas9 plasmids for obtaining deletions of the H19- and IG-DMRs. To exclude diploid ESCs and isolate only haploid ESCs expressing EGFP, a specific sort gate was defined (Figure 2A). Single haploid EGFP-positive cells were then plated into individual wells of 96-well plates to obtain sub-clones. MEF feeders were used to increase the plating efficiency and survival of the transfected phaESCs. After expansion of the cultures, an initial round of genotyping was carried out by PCR to identify sub-clones that carry deletions of both DMRs. After MEF feeders had been removed from the cultures, a second genotyping was performed to confirm the absence of wildtype alleles at the H19- and IG-DMRs (Figure 2B). From a total of 135 sub-clones, we obtained 5 haploid DKO-phESC lines that carried the deleted alleles and were free of wildtype alleles of both the H19-DMR and the IG-DMR.
A CAG-EGFP transgene was introduced into DKO-phaESCs to study their contribution to semi-cloned embryos by visualization of green fluorescence under a microscope (Figure 3A). For the intracytoplasmic injection, DKO-phaESCs were treated with demecolcine to arrest them in M-phase. Thus, the cell cycle of DKO-phaESCs was synchronized with that of MII oocytes. Flow cytometry analysis showed 2 populations corresponding to G2/M phase arrested haploid (2n) and diploid cells (4n) after the treatment with demecolcine (Figure 3B). Absence of the 1n haploid peak indicated that the cell cycle arrest was largely complete. M-phase haploid ESCs were then sorted and injected into oocytes. For this, a single DKO-phaESC was loaded into the microinjection pipette and injected into the cytoplasm of a MII oocyte (Figure 3C). The plasma membrane of the DKO-phaESC was ruptured by pipetting into the tip of the microinjection pipette.
After the injection, EGFP expression was rarely detected in the constructed semi-cloned embryos as the cytoplasm of DKO-phaESCs had dispersed in the large cytoplasm of the oocyte (Figure 3D). In rare cases, a round spot of intense EGFP expression could be observed within the ooplasm. This observation was likely caused by the inadvertent injection of intact DKO-phaESCs. Failure to rupture the DKO-phaESC cell membrane is likely not compatible with further embryo development and should be avoided. One hour after the injection, embryos were activated by treatment with strontium chloride18. Six hours after the initiation of activation, up to 3 polar bodies were observed under the microscope (Figure 3E). These polar bodies correspond to the first and second polar bodies of the oocyte and a pseudo polar body from the DKO-phaESC7. In addition, two pronuclei were observed under a differential interference contrast microscope, which resembled the pronuclear stage of zygotes after normal fertilization with sperm.
To demonstrate developmental competence, semi-cloned embryos were cultured to the blastocyst stage (Figure 5A). Furthermore, a full-term mouse was obtained after transferring semi-cloned 2-cell stage embryos into the oviducts of a recipient female (Figure 5B). As expected, the mouse derived from a semi-cloned embryo was a female as neither oocytes nor oocyte-derived phaESCs carry a Y chromosome. The semi-cloned mouse was overtly normal and produced healthy offspring when mated with a wildtype Swiss Webster male. Until now, the semi-cloned mouse has been alive for over 600 days without any apparent health problems.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61999/61999fig01v3.jpg" /> 
Figure 1: Overview of the application of DKO-phaESCs as sperm replacement.&#160;(A) A time frame of the procedures of the protocol. (B) The scheme shows the steps to establish DKO-phaESC lines (steps 1&#8211;6). (C) The scheme shows the steps to construct semi-cloned embryos by intracytoplasmic injection of a DKO-phaESC into a MII oocyte (steps 7&#8211;14). Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell; FACS = fluorescence-activated cell sorting; MEF = mouse embryonic fibroblast. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61999/61999fig02v2.jpg" /> 
Figure 2: Establishment of DKO-phaESC lines by CRISPR/Cas9-mediated deletions of H19- and IG-DMRs.&#160;(A) Flow cytometry analysis of phaESCs after transfection with piggyBac plasmids for stable EGFP expression and with 4 CRISPR/Cas9 plasmids. Non-transfected phaESCs are shown as control. The DNA profile (top) shows the cell cycle distribution of haploid and diploid cells. G1/S-phase haploid cells expressing EGFP are indicated by the green gate (bottom, right). (B) Genotyping of phaESC sub-clones that were grown on MEFs (first genotyping) and after removal of MEFs (second genotyping). Sub-cloned phaESC lines 1, 2, 3, and 4 represent wildtype cells, cells with a H19-DMR deletion, with an IG-DMR deletion, and with combined H19- and IG-DMR deletions, respectively. DKO-phaESC lines 5&#8211;8 possess both H19-DMR and IG-DMR deletions and are free from wildtype alleles. Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell; DMR = differentially methylated region; MEF = mouse embryonic fibroblast; EGFP = enhanced green fluorescent protein; WT = wildtype. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61999/61999fig03v3.jpg" /> 
Figure 3: Injection of mitotically arrested DKO-phaESCs into MII oocytes.&#160;(A) Morphology of a DKO-phaESC culture that carries a CAG-EGFP transgene and deletions of the H19- and IG-DMRs; scale bar = 50 &#181;m. (B) Representative flow cytometry analysis of DKO-phaESCs after arrest with demecolcine for 8 h. DKO-phaESCs without demecolcine treatment are shown as control. (C) Morphology of DKO-phaESCs in the microinjection pipette. A single intact DKO-phaESC before (left) and after (right) rupturing the plasma membrane by pipetting is shown; scale bar = 20 &#181;m. (D) Constructed embryos at 1 h after injection of DKO-phaESCs into MII oocytes. A merged image of EGFP fluorescence and bright field is shown. Black arrowheads indicate round spots of intense EGFP expression after injection of intact DKO-phaESCs, which should be avoided; scale bar = 50 &#181;m. (E) A differential interference contrast image of semi-cloned embryos 6 h after the initiation of activation with strontium chloride is shown. White arrowheads indicate embryos with 3 polar bodies including one pseudo polar body from the injected DKO-phaESC; scale bar = 50 &#181;m. Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell; EGFP = enhanced green fluorescent protein. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61999/61999fig04.jpg" /> 
Figure 4: Scheme of the setup for intracytoplasmic injection of DKO-phaESCs into oocytes.&#160;(A) The arrangement of an injection pipette, a holding pipette, and an oocyte in the injection chamber is shown. &#945;, bend&#160;angle of the microinjection pipette. (B) Layout of the drops in the micromanipulation dish for intracytoplasmic injection. Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61999/61999fig05.jpg" /> 
Figure 5: Development of semi-cloned embryos.&#160;(A) Preimplantation development of semi-cloned embryos in vitro. EGFP expression is initially observed in four-cell embryos at day 2 after intracytoplasmic injection. At day 4, intense EGFP expression can be observed in blastocysts; scale bar = 100 &#181;m. (B) A semi-cloned mouse obtained after 2-cell embryo transfer to a recipient female. At 74 dpp, the semi-cloned mouse (arrowhead) delivered her first pups (asterisk) by natural birth after mating with a wildtype Swiss Webster male. Semi-cloned embryos and the semi-cloned mouse shown in this figure are identical with ones reported in Aizawa et al.3. Abbreviations: EGFP = enhanced green fluorescent protein; .dpp, days post-partum. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Gelatin solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>500 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>-</td>
			<td>1 g</td>
			<td>0.2%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Haploid embryonic stem cell (HaESC) medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>NDiff 227</td>
			<td>-</td>
			<td>10 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>10 mM</td>
			<td>3 &#181;L</td>
			<td>3 &#181;M</td>
		</tr>
		<tr>
			<td>PD 0325901</td>
			<td>10 mM</td>
			<td>1 &#181;L</td>
			<td>1 &#181;M</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>1 x 106 IU/mL</td>
			<td>10 &#181;L</td>
			<td>1,000 IU/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100x</td>
			<td>100 &#181;L</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">HaESC maintenance buffer</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>HaESC medium</td>
			<td>-</td>
			<td>1 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>1 M</td>
			<td>20 &#181;L</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Wash buffer</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>DMEM / F-12</td>
			<td>-</td>
			<td>100 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BSA fraction</td>
			<td>7.5%</td>
			<td>7.1 mL</td>
			<td>0.5%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">MEF medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>-</td>
			<td>500 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>-</td>
			<td>56 mL</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>14.3 mol/L</td>
			<td>3.9 &#181;L</td>
			<td>100 &#181;M</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100x</td>
			<td>5.6 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Lysis buffer</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>8.25 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 8.5)</td>
			<td>1 M</td>
			<td>1 mL</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0.5 M</td>
			<td>100 &#181;L</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>SDS solution</td>
			<td>10%</td>
			<td>200 &#181;L</td>
			<td>0.2%</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 M</td>
			<td>400 &#181;L</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>20 mg/mL</td>
			<td>50 &#181;L</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Activation medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>-</td>
			<td>1 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Strontium chloride</td>
			<td>1 M</td>
			<td>5 &#181;L</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.5 M, pH 8.0</td>
			<td>4 &#181;L</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">PVP solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>-</td>
			<td>5 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PVP</td>
			<td>-</td>
			<td>0.6 g</td>
			<td>12%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">PMSG solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>-</td>
			<td>450 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>500 IU/mL</td>
			<td>50 &#181;L</td>
			<td>50 IU/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">hCG solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>-</td>
			<td>450 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>500 IU/mL</td>
			<td>50 &#181;L</td>
			<td>50 IU/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Hyaluronidase medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>-</td>
			<td>380 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>10 mg/mL</td>
			<td>20 &#181;L</td>
			<td>0.5 mg/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Abbreviations: LIF = leukemia inhibitory factor; MEF = mouse embryonic fibroblast;</td>
		</tr>
		<tr>
			<td colspan="4">FBS = fetal bovine serum; DMEM = Dulbecco&#39;s Modified Eagle Medium; BSA = bovine serum albumin;</td>
		</tr>
		<tr>
			<td colspan="4">EDTA = ethylenediaminetetraacetic acid; SDS = sodium dodecylsulfate; EGTA = ethylene-bis(oxyethylenenitrilo)tetraacetic acid;</td>
		</tr>
		<tr>
			<td colspan="4">PBS = phosphate-buffered saline; PVP = polyvinylpyrrolidone; hCG = human chorionic gonadotropin;</td>
		</tr>
		<tr>
			<td colspan="4">PMSG = pregnant mare serum gonadotropin.</td>
		</tr>
	</tbody>
</table>
Table 1: Recipe of medium, buffer, and solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td colspan="2">Sequence (5&#39; to 3&#39;)</td>
			<td>Application</td>
		</tr>
		<tr>
			<td>H19-DMR-P1</td>
			<td colspan="2">GTG GTT AGT TCT ATA TGG GG</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>H19-DMR-P2</td>
			<td colspan="2">AGA TGG GGT CAT TCT TTT CC</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>H19-DMR-P3</td>
			<td colspan="2">TCT TAC AGT CTG GTC TTG GT</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>IG-DMR-P1</td>
			<td colspan="2">TGT GCA GCA GCA AAG CTA AG</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>IG-DMR-P2</td>
			<td colspan="2">CCA CAA AAA CCT CCC TTT CA</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>IG-DMR-P3</td>
			<td colspan="2">ATA CGA TAC GGC AAC CAA CG</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA1-F</td>
			<td colspan="2">CAC CCA TGA ACT CAG AAG AGA CTG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA1-R</td>
			<td colspan="2">AAA CCA GTC TCT TCT GAG TTC ATG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA2-F</td>
			<td colspan="2">CAC CAG GTG AGA ACC ACT GCT GAG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA2-R</td>
			<td colspan="2">AAA CCT CAG CAG TGG TTC TCA CCT</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA1-F</td>
			<td colspan="2">CAC CCG TAC AGA GCT CCA TGG CAC</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA1-R</td>
			<td colspan="2">AAA CGT GCC ATG GAG CTC TGT ACG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA2-F</td>
			<td colspan="2">CAC CCT GCT TAG AGG TAC TAC GCT</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA2-R</td>
			<td colspan="2">AAA CAG CGT AGT ACC TCT AAG CAG</td>
			<td>gRNA</td>
		</tr>
	</tbody>
</table>
Table 2: List of oligonucleotides.

Watch this Scientific Journal Video about Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm at JoVE.com

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

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 ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

4.5K Views.  ETH Zurich. 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.

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

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

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

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.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. ...

Genetics

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

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the efficiency in developing gene-knockout mice by inducing small indel mutation would be sufficient enough, the efficiency of embryo genome editing for making large-size DNA knock-in (KI) is still low. Therefore, in contrast to the direct KI method in embryos, gene targeting using embryonic stem cells (ESCs) followed by embryo injection to develop chimera mice still has several advantages (e.g., high throughput targeting in vitro, multi-allele manipulation, and Cre and flox gene manipulation can be carried out in a short period). In addition, strains with difficult-to-handle embryos in vitro, such as BALB/c, can also be used for ESC targeting. This protocol describes the optimized method for large-size DNA (several kb) KI in ESCs by applying CRISPR/Cas9-mediated genome editing followed by chimera mice production to develop gene-manipulated mouse models.

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.

The CRISPR/Cas9 system has made it possible to develop genetically modified mice by direct genome editing using fertilized zygotes. However, although the ...

Murine Round Spermatid Injection Procedure to Study Embryonic Development

Mouse Round Spermatid Injection

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of round spermatid injection (ROSI), oocytes can be successfully fertilized and developed into viable fetuses. In a groundbreaking milestone achieved in 1995, the first mouse fetus was born through ROSI technology. ROSI has since emerged as a pivotal tool for unraveling the intricate mechanisms governing embryonic development and holds significant potential in various applications, including the acceleration of mouse generation and the production of genetically modified mice. In 1996, a milestone was reached when the first human fetus was born through ROSI technology. However, the clinical applications of this method have shown a fluctuating pattern of success and failure. To date, ROSI technology has not found widespread application in clinical practice, primarily due to its low birth efficiency and insufficient validation of fetal safety. This article provides a comprehensive account of the precise methods of performing ROSI in mice, aiming to shed new light on basic research and its potential clinical applications.

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.

Round spermatids, characterized by their haploid genetic content, represent the precursor cells to mature spermatozoa. Through the innovative technique of ...

ICSI-ET Mouse Model to Study Long-Term Impacts on Glucose Metabolism

Monitoring Blood Glucose in Mouse Offspring After Intracytoplasmic Sperm Injection

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life roughly equivalent to 40 human years. Intracytoplasmic sperm injection (ICSI) is a commonly used assisted reproductive technology in clinical practice. However, given its relatively recent emergence about 30 years ago, the long-term effects of this technique on human development remain unclear. In this study, we established the ICSI combined with embryo transfer (ET) method using a mouse model. The results demonstrated that normal mouse sperm, after undergoing in vitro culture and subsequent ICSI, exhibited a fertilization rate of 89.57% and a two-cell rate of 87.38%. Following ET, the birth rate of offspring was approximately 42.50%. Furthermore, as the mice aged, fluctuations in glucose metabolism levels were observed, which may be associated with the application of the ICSI technique. These findings signify that the mouse ICSI-ET technique provides a valuable platform for evaluating the impact of sperm abnormalities on embryo development and their long-term effects on offspring health, particularly concerning glucose metabolism. This study provides important insights for further research on the potential effects of the ICSI technique on human development, emphasizing the necessity for in-depth investigation into the long-term implications of this technology.

Author Spotlight: Exploring the Long-Term Health Impacts of Intracytoplasmic Sperm Injection on Offspring

Here, we present a protocol to establish an intracytoplasmic sperm injection (ICSI)-embryo transfer (ET) mouse model, allowing us to observe age-related changes in glucose metabolism that may be attributed to ICSI, providing insights into its potential long-term impacts on human development.

Human lifespan is considerably long, while mouse models can simulate the entire human lifespan in a relatively short period, with one year of mouse life ...

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 ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

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 was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...

Biology

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

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;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&#x2022;Cryo kit and later successfully recovered by IVF.

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

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.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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

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.

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.

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...