Name: Nuclear Transfer into Mouse Oocytes (Video) | JoVE Journal
Uploaded: 2006-11-30T00:00:00.000+00:00
Duration: 14 min 17 s
Description: Learn nuclear transfer techniques for reprogramming differentiated nuclei into embryonic states using mouse oocytes. This protocol demonstrates essential reagents and optimal conditions for successful cloning and stem cell research applications.

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5 minutes

DE would like to thank Dr. Hide Akutsu for sharing his NT tricks and Dr. Stephen Sullivan and Garrett Birkhoff for critical reading of the protocol.

Preparations:
<ol>
<li class="jove_step">A detailed description of superovulation and microdrop embryo culture can be found elsewhere 2. </li>
<li class="jove_step">Prepare a petri dish with microdrops of embryo culture medium (e.g. KSOM, Chemicon). Cover with mineral oil (mineral oil batches should be tested for compatibility with embryo culture). Equilibrate at 37&deg;C in air plus 5% CO2. (In Thermo-Electron 3110 water jacketed incubator or equivalent)</li>
<li class="jove_step">Prepare a dish for the isolation of oocytes: Place drops of hepes buffered CZB &amp; hyaluronidase (0.1% w/v, Sigma) in one half of the dish and HCZB in the other half. Cover with mineral oil. Keep the dish warm on a heated stage of a dissection microscope.</li>
<li class="jove_step">Prepare the microscope: Place a holding pipette of an outer diameter of about 50&micro;m&nbsp; (slightly smaller than the diameter of the oocyte), and an opening of about 20&micro;m in one pipette holder. Holding pipettes are easy to make with the necessary equipment, a needle puller (Sutter Instruments) and a microforge (Narishige), but they may also be purchased from Humagen.&nbsp; Load 3-5 &micro;l mercury into the tail of a micropipette (inner diameter 8&micro;m). Avoid mercury spills. Prepare a dish with several small drops each of 11% w/v PVP (polyvinylpyrrolidone) in HCZB and 5&micro;g/ml cytochalasin B in HCZB.&nbsp; Use the lid of a Petri dish rather than the dish itself as the lid has a short rim that will not interfere with the movement of the holding and the enucleation pipettes. Align the holding pipette and the enucleation pipette in an HCZB drop. Aspirate a little HZCB into the tip of the holding pipette. </li>
<li class="jove_step">Preparation of donor cells depends largely on the cell type and the experiment the investigator intends to perform. In general, donor cells should be treated with a proteolytic enzyme such as trypsin and kept on ice to reduce stickiness until transfer. </li>
<li class="jove_step">Humanely euthanize mice 14-15 hours after the administration of hCG. Harvest oviducts and dissect them in the warm HCZB-hyaluronidase drops. After approx. 5 min, oocytes will be mostly free from cumulus cells. They should then be washed several times in HCZB, then washed further in the pre-equilibrated KSOM drops, and kept in the incubator until enucleation.</li>
</ol>
Enucleation
Manipulations are done with hydraulic micromanipulators, (Narishige) on an inverted microscope, such as the Nikon TE200. A piezo micromanipulator (Primetech) is used for drilling the zona pellucida. Flat-tipped enucleation and transfer pipettes can be purchased from Humagen. Clean and lubricate the enucleation pipette in the PVP drops by expelling microscopic mercury droplets and by aspirating and expelling PVP. To prevent stickiness of the needle, the same procedure should be done between each group of oocytes that are either enucleated or transferred.
Place a small group of oocytes into the HCZB-cytochalasin B drop. The size of the group should be adapted to the level of experience of the investigator. Oocytes should not be kept on the stage for longer than 20-30 min. Move the instruments to the HCZB-cytochalasin B drops. The holding pipette and the enucleation pipette should be brought into alignment with the equatorial plane of the oocyte. This will be possible if the holding pipette has an outer diameter that is slightly smaller than the oocyte itself. Rotate an oocyte until the differently refractive metaphase spindle can be seen. If a beginner cannot see the spindle, the metaphase plate can be identified using the DNA dye Hoechst and UV illumination, however, that is not compatible with efficient embryonic development. Position the metaphase spindle at 3 o&rsquo;clock and hold it well with the holding pipette. Apply piezo pulses to penetrate the zona pellucida. Touch the metaphase plate with the enucleation pipette. Once the resistance of the metaphase spindle can be &lsquo;felt&rsquo;, aspirate the spindle and withdraw the needle. The oocyte cytoplasm is fluid, the metaphase spindle however moves as a clump when touched with the pipette. Try to reduce the amount of cytoplasm that is removed with the spindle. After a group of oocytes has been enucleated, wash them through embryo culture medium and place them into the incubator until transfer. Some enucleated oocytes should not be transferred but should be activated and serve as a control for enucleation. These oocytes will fragment within several hours, indicating successful enucleation.
Nuclear transfer
Place cells into PVP solution (11% PVP in HCZB). If many clones arrest at the 1-cell stage, the PVP concentration should be lowered to 5% or less. Mix cells well with PVP. Pick up cells with a pipette with an inner diameter slightly smaller than the diameter of the cell. The membrane of the cell should break upon aspiration. In some cases, repetitive expelling and aspiration as well as the application of gentle piezo pulses can be used to break the donor cell. A new drop of donor cells should be made with each group of oocytes being transferred, as donor cells become sticky after some time. Inject the broken donor cell shortly after breaking the membrane. For the transfer, focus on the equatorial plane of the oocyte itself rather than the equatorial plane of the zona pellucida, and position the transfer and the holding pipette in the same plane. Hold the oocyte including a part of its cytoplasm firmly. Penetrate the zona pellucida using piezo pulses. Bring the donor nucleus to the tip of the pipette, then push the pipette tip almost to the opposite side of the enucleated oocyte, close to the holding pipette, making a deep furrow in the oocyte. Aspirate a very small amount of oocyte cytoplasm into the pipette, apply a single weak piezo pulse to break the oocyte membrane, eject the donor nucleus, withdraw the needle rapidly while aspirating at the cytoplasmic membrane at the right end of the furrow. By simultaneously withdrawing the needle and aspirating, it should be possible to close the hole left behind by NT. This 'hole removal technique will reduce the number of oocytes that lyse during NT. Wash the reconstructed oocytes in KSOM and return them to the incubator for 1-3 hours.
Oocyte activation
Prepare one half of a dish with droplets of calcium free MCZB and the other half with droplets of calcium free MCZB plus 10mM Sr2+ and 5&micro;g/ml cytochalasin B to inhibit polar body extrusion. Equilibrate for 30 min. under 5% CO2 in air. Wash NT embryos well in calcium-free MCZB and then place them in small groups in different drops of calcium-free MCZB.&nbsp; Return the dish to the incubator and culture for 5-6 hours. To control for the artificial activation protocol and for embryo culture conditions, non-enucleated oocytes should be used. They will develop as parthenogenetic embryos. Following activation, wash the embryos through KSOM and place embryos in the incubator for long-term culture. Pronuclei should be visible at this time point, indicating successful transfer.
Embryo Culture Media Cookbook
Master Salts
Begin with 980 mL ultrapure H2O in sterile 1 L bottle
Add dry components:
NaCl&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 4760 mg (81mM)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;Sigma S-5886 KCl&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;360 mg (5mM)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma P-5405 MgSO4 &bull; 7H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 290 mg (1.18mM)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma M-2773 KH2PO4&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 160 mg (1.17mM)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Sigma P-5655 EDTA 2NA&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 40 mg (0.1mM)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma E-6635 Glucose (D)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1000 mg (5.5mM)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Sigma G-6152 &nbsp;
Add liquid Components
Na-lactate (lactic acid)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 5.3 mL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;&nbsp;&nbsp; Sigma 44263 Pen&rsquo;Strep&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 10 mL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Gibco 15140-122
For MCZB Stock Salts:
Filter sterilize 500 mL master salts&nbsp; (good for 3-4 months; store at 4&deg; C)
&nbsp;
For HCZB Stock Salts:
Start with 500 mL master salts
Add 50 mg PVA (cold-soluble; Sigma P-8136)
Stir for 30-60 min and sterile filter (good for 3 months; store at 4&deg; C)
&nbsp;
Ca++ Free MCZB (for oocytes activation in 5% CO2)
Start with 99 mL MCZB stock salts
Add dry components:
 NaHCO3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 211 mg&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma S-5761 &nbsp; Na-pyruvate (pyruvic acid)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;3 mg&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Sigma P-4562 &nbsp; L-Glutamine&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 15mg&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma G-8540 &nbsp; Bovine serum albumin (BSA)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 500 mg&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma A-3311
Swirl until all components are dissolved; sterile filter.
&nbsp;
HCZB (for micromanipulation in ambient atmosphere)
Start with 99 mL HCZB stock salts
Add dry components:
 Hepes-Na&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;520 mg&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Sigma H-3784 &nbsp; NaHCO3&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;42 mg&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; &nbsp;Sigma S-5761 &nbsp;
Add liquid components:
&nbsp;
128 mM CaCl2&nbsp; (18.8g/l)&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; 1 mL&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Sigma C-7902
&nbsp;
Adjust pH to 7.5 with 1 N HCl
Swirl until dissolved; sterile filter.

<ol><li>Eggan, K., Jaenisch, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aEggan%2C+K+%22Mammalian+and+Avian+Transgenesis-+New+Approaches%22">Mammalian and Avian Transgenesis- New Approaches</a>. Pease, S., Lois, C. , Springer. (2006).</li>
<li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aNagy%2C+A+%22Manipulating+the+mouse+embryo%22">Manipulating the mouse embryo</a>. Press, C. S. H. L. , (2003).</li>
<li>Wakayama, T., Perry, A. C., Zuccotti, M., Johnson, K. R., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Full-term+development+of+mice+from+enucleated+oocytes+injected+with+cumulus+cell+nuclei%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei.</a> Nature. 394, 369-374 (1998).</li>
</ol>

The authors have nothing to disclose.

<table><tbody><tr><td>Cytochalastin B</td><td>Sigma-Aldrich</td><td></td><td>100x stock = 500 &amp;#181;g/ml Cytochalasin B in DMSO. Store at -20 &amp;#730;C.</td></tr><tr><td>Strontium Chloride </td><td></td><td></td><td>10x stock = 100 mM SrCl&lt;sub&gt;2&lt;/sub&gt; in H&lt;sub&gt;2&lt;/sub&gt;O. Store at room temperature.</td></tr><tr><td>MCZB Stock Salts</td><td></td><td></td><td>Filter sterilize 500 ml master salts (good for 3-4 months; store at 4 &amp;#730;C)</td></tr><tr><td>HCZB Stock Salts</td><td></td><td></td><td>Start with 500 mL master salts. Add 50 mg PVA. Stir for 30-60 min and sterile filter (good for 3 months; store at 4 &amp;#730;C).</td></tr><tr><td>PVA</td><td>Sigma-Aldrich</td><td>P-8136</td><td>cold-soluble</td></tr><tr><td>HCZB with 11% w/v PVP</td><td></td><td></td><td>Start with 20 ml HCZB medium in a 50 ml conical tube. Place 22 g PVP on top of liquid. Close tube and store undisturbed at 4 &amp;#730;C for 72 &amp;#8211; 96 hr, at which time the PVP will have entered solution. Filter sterilize through an 8 mm filter and store at 4 &amp;#730;C.</td></tr><tr><td>PVP</td><td>ICN Biomedicals</td><td></td><td>MW 360,000</td></tr><tr><td colspan="4">Please check for additional buffers compositions in the Protocol part.</td></tr></tbody></table>

nuclear transfer into mouse oocytes

Nuclear Transfer into Mouse Oocytes

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying development, differentiation and aging are epigenetic rather than genetic processes. The reversibility of these processes opens exciting perspectives in basic research, and in the more distant future, in regenerative medicine. In the mouse, embryonic stem cells can be derived from cloned preimplantation stage embryos. Such embryonic stem cells have the ability to give rise to all cell types of the adult organism. Importantly, these cells are genetically identical to the donor. If applicable to human, this would allow the derivation of stem cells from a patient. These cells could then be differentiated into the affected cell type of the patient and studied in vitro, or used to replace the damaged or missing cells. The study of nuclear transfer in the mouse remains important as it can inform us about the principles of nuclear reprogramming. This movie and the accompanying protocol are intended to help learning nuclear transfer in the mouse, a method initially developed in the group of Prof. Yanagimachi (WAKAYAMA et al. 1998).

This movie and the protocol are intended to help learning nuclear transfer.

Nuclear transfer into an unfertilized oocyte can restore developmental potential to a differentiated cell. This demonstrates that the processes underlying ...

So we do NU Nuclear transfer because our group is interested in reprogramming. This is a fascinating process in that the differentiated nucleus can be reprogrammed into an embryonic state. These are the reagents case SUM and HCCV and this is hyaluronidase.And first I prepare a dish with, and this will go to the incubators to equate with a CO2. These are drops, are several drops for Turing that covered with mineral oil. So they calibrate CO2 atmosphere.Now make drop of HCZB. This will be used for washing. This is high on days and again covered in this mineral oil.This high, this drops with hyaluronidase. This part here are just HCCP drops. These are for washing.This is for removing ulu cells from the O side. This stage is heated so it will warm up until I come back and get the oil signs from the mice and cover everything with mineral oil. This here are PVP drops for cleaning of the needle and for putting the cells into, and this is HCCB plus Cyla B.And here I will make the manipulations.Okay, this here is a needle color. What I'm doing now is I pull a needle. Alright, now I have a very nice needle, very thin.And now this is a micro forge. I have to cut the needle at the certain position to have the right size holding pipette. I heat the glass ball, I go down with the needle melts to it, and now it's broken.I push this away. You see the broken needle is perfect. Now I go down here, I push up the heat.Now the glass ball will turn red, it will melt, push the glass ball and it, it makes a curve. That's enough. I remove it.You see, then I insert the needle. We just made the holding pipette sub it into this head. Now insert it back into the microscope.Micro pipette, capillary and I loaded with mercury. I loaded it into this tip. Mercury load this capillary here with mercury.It goes inside. Set the needle here in this head. Screw tight, pull it back.Turning clockwise here will push the mercury here to the front so you can see it. And now the tip into the oil.See? So now I'm moving down the needle to get the vision.Expel some mercury droplets and suck up some QVP. That's all right. And then move it up again.I go to the HCCB and move down the hole. My head, I go to P, which is the mouse pipette. I need to prepare a capillary to insert it here.Thanks.Already.Good.So make some capillary.Okay. And in the pipe. I am ley.I'm coming from Switzerland and I started this work one year ago and I came to K'S lab and I started learning nuclear transfer. It's really, it's a great place here and it's a great to work here. And, and of course this is a very complex experiment and many problems may arise while doing it.So when you start, you may encounter problems like many cells lysing while you either nucleate or while you do the transfer. Or then you find even though you transfer successfully, they don't develop. And there are always, can be numerous reasons.I mean you can start from the culture system of the all sites. So one always needs some controls like the parts of the genetic control or some fertilized emperors to test the culture system, the incubators. And then you also need to improve the, the skills of the microscope.So it just takes a lot of practice until you reliably can inate the side and then transfer back. This is just a matter of practice and we'll take a few weeks or or months until you are good at it. And otherwise it's ready for lengthy experiments.So it's good to reserve a whole day for it. And then something tricky in the beginning is also the mouse pipetting. So not to make bubbles to, to control it well and not to lose the oversight or especially you can also damage a clone, particularly because the outer layer, the so of lucita is open, it's actually possible when you mouth, you damage that clone and it kind of leaks out of that sonar lucita.So when also needs to be careful not to have oil in that mouth pipe. Not to suck too much and not to blow too much, but this is relatively easily acquired, this skill.

Research

JoVE Journal

Biology

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation.  Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome.  Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

This video describes the pronuclear injection of lentiviral vectors into the perivitelline space of a fertilized mouse embryo. The lentivirus contains the transgene, which is incorporated into the genome of the fertilized embryo, eventually giving rise to transgenic mice.

Lentiviral Vector-Based Perivitelline Injection: A Method to Transduce the Gene of Interest Into Fertilized Single-Cell Mouse Oocytes

Here we present a protocol for the characterization of mouse antral oocytes based on nucleolar organization.

Isolation and Characterization of Mouse Antral Oocytes Based on Nucleolar Chromatin Organization

We describe a dramatically improved method for mouse cloning using trichostatin A, vitamin C, and deionized bovine serum albumin. We show a simplified, reproducible protocol that supports efficient development of cloned embryos. Hence, this method could become a standardized procedure for mouse cloning.

Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer

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.

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

An optimized protocol is presented that enables the depletion of cytoplasmic microtubule organizing centers in mouse oocytes during metaphase I using a near-infrared femtosecond laser.

Multi-Photon Laser Ablation of Cytoplasmic Microtubule Organizing Centers in Mouse Oocytes

Error in chromosome segregation is a common feature in oocytes. Therefore, studying the spindle assembly checkpoint gives important clues about the mechanisms needed to produce healthy eggs. The present protocol describes three complementary assays to evaluate spindle assembly checkpoint integrity in mouse oocytes.

Evaluation of the Spindle Assembly Checkpoint Integrity in Mouse Oocytes

This protocol provides an experimental framework to document the physical impact of the cytoskeleton on nuclear shape and the internal membrane-less organelles in the mouse oocyte system. The framework can be adapted for use in other cell types and contexts.

Capturing Cytoskeleton-Based Agitation of the Mouse Oocyte Nucleus Across Spatial Scales

This protocol illustrates the technique for extracting oocytes or early-stage fertilized embryos from the oviduct of mice. The ability to identify the infindibulum and insert a blunt end needle into it is essential to correctly performing the procedure.

Retrieval of Mouse Oocytes

Learn nuclear transfer techniques for reprogramming differentiated nuclei into embryonic states using mouse oocytes. This protocol demonstrates essential reagents and optimal conditions for successful cloning and stem cell research applications.

Nuclear Transfer into Mouse Oocytes

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