Name: transnuclear mice with pre-defined t cell receptor specificities against toxoplasma gondii obtained via scnt
Uploaded: 2010-09-30T17:00:00
Duration: 13 min 36 s
Description: Watch this Scientific Journal Video about Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT at JoVE.com

The authors are thankful to H. Eisen, M. Gubbels, K. van Grinsven, E. Guillen, A. Drake, V. Mahajan, Q. Gao, and G. Yap for fruitful discussions and reagents. We are thankful to J. Dausman, R. Flannery, and J. Jackson for assistance with the management of the mouse colony. We are thankful to P. Wisniewski for FACSorting. EMF was supported by the Human Frontiers Science Program. R.J. was supported by National Institute of Health grants RO1-HD045022 and R37-CA084198. HLP was supported by grants from the National Institute of Health.

This method was used in the research reported in <a href="http://www.sciencemag.org/cgi/content/abstract/328/5975/243">Kirak et al., Science 328 (5975), 243-248 (2010)</a>.
1. Isolation of Donor Cells
Before specific T or B cells can be used as donor cells to generate transnuclear mouse models, mice need to be infected with a pathogen of interest or immunized with an antigen of interest. Since we have used CD8+ cells specific for Toxoplasma gondii, the following protocol will describe the generation and isolation of these cells and needs to be adapted according to personal interest.
<ol>
 <li>Cysts derived from a mouse brain homogenate (about 40) are injected intra-peritoneally into BL/6 x Balb/c F1 (B6CF1) mice, which enables isolation of both H-2b and H-2d restricted CD8+ T cells.</li>
 <li>At the peak of the immune response, the infected mouse is euthanized, the spleen isolated and placed into a dish containing 6 mL red blood cell (RBC) lysis buffer.</li>
 <li>Using the frosted sides of two cover slides, the spleen is carefully ground and incubated in the RBC lysis buffer for 5 min at RT.</li>
 <li>Add 14 mL PBS, filter the cell suspension through a cell strainer (40 or 70 &mu;m) into a 50 mL falcon tube and centrifuge for 5 min at 300g.</li>
 <li>Remove supernatant, resuspend the cell pellet in 1 mL PBS and transfer into a 1.5 mL Eppendorf tube. Centrifuge for 5 min at 300g and remove the supernatant afterwards.</li>
 <li>Resuspend the cell pellet in 50 &mu;L PBS, add fluorescently labeled antibodies to identify the cell type of interest (e.g. CD3, B220, CD8, and CD4), and fluorescently labeled MHC-I tetramer (MHC-II tetramer for CD4 T cells) loaded with the peptide of interest to the cell suspension and incubate for 30 min at 4&deg;C.</li>
 <li>Add 1 mL PBS, centrifuge for 5 min at 300 g, resuspend pellet in 3 ml PBS, and filter the cell suspension through a cell strainer (40 or 70 &mu;m) into a tube.</li>
 <li>Perform FACSorting by setting the gates stringently (Figure 1B).</li>
</ol>
2. Somatic Cell Nuclear Transfer
There are a few protocols for Somatic Cell Nuclear Transfer. The one described here makes use of oocytes arrested at Metaphase-II and the inhibition of the second meiotic division using Cytochalasin B. Therefore donor cells are needed which are in G0/G1 phase.
<ol>
 <li>Preparation of oocytes
 <ol>
 <li>Female mice of BDF1 background are injected intra-peritoneally with 5IU PMS per mouse between 5PM and 7PM.</li>
 <li>About 47 hours later, each mouse is injected intra-peritoneally with 5IU HCG.</li>
 <li>Prepare a culture dish (KSOM-AA drops under oil) for the oocytes on the same day as the HCG injection and place them into an incubator 37&deg;C, 5% CO2. Also, prepare the needles necessary for SCNT (7&mu;m for enucleation and 4 or 5&mu;m for nuclear transfer of lymphocyte nuclei) by loading them with mercury.</li>
 <li>Euthanize mice and isolate oviducts about 13h after HCG (approximately 8AM). Collect the oviducts in 1-2 drops of HCZB.</li>
 <li>One-by-one move the oviducts into a drop containing M2 w/Hyaluronidase. Nick the oviduct carefully with a forcep and move the oocyte-cumulus-complex into the drop. After all oocyte-cumulus-complexes have been isolated, the dish is incubated at 37&deg;C.</li>
 <li>After 2-5 min (the exact time depends on the batch of Hyaluronidase) collect the oocytes, wash in HCZB and plate them into KSOM-AA drops.</li>
 </ol>
 </li>
 <li>Enucleation of oocytes
 <ol>
 <li>Use the lid of a petri dish to prepare the SCNT plate. Set up the microscope, and the micromanipulator.</li>
 <li>Wash the enucleation needle (7 &mu;m) in PVP before starting with enucleation.</li>
 <li>For enucleation, place a group of oocytes (10-30) into a drop of HZCB with Cytochalasin B (5 &mu;g/mL). While incubating (min. 5 min) line-up the oocytes horizontally.</li>
 <li>Take one oocyte and place it in front of the holding needle. Fix the oocyte to the holding needle by applying a vacuum. Using the enucleation needle, turn the oocyte until the chromosome-spindle-complex (CSC, often called the "nucleus") is in the 3 o'clock position.</li>
 <li>Using piezo-pulse penetrate the zona pellucida carefully without damaging the oocyte. Place the opening of the needle adjacent to the Metaphase-II spindle and apply vacuum. The "nucleus" should slowly move into the needle, and the membrane should seal itself once it is removed completely.</li>
 <li>Transfer the enucleated oocytes back into the KSOM-AA drops, and wash them multiple times. Repeat the enucleation steps with a new group of oocytes. Continue until all eggs are enucleated or until about 11AM.</li>
 </ol>
 </li>
 <li>Nuclear Transfer
 <ol>
 <li>For the nuclear transfer, exchange the enucleation needle (7 &mu;m) with a nuclear transfer needle (4 or 5 &mu;m) and wash it with PVP.</li>
 <li>Place your cells of interest (e.g. CD8+ T cells) into a drop with PVP, mix well and incubate for a minimum of 10 min.</li>
 <li>Place a group of enucleated oocytes (10-30) into a drop of HCZB with Cytochalasin B (2.5 &mu;g/mL). While incubating (min. 5 min) line-up oocytes horizontally by using the nuclear transfer needle.</li>
 <li>Pick up a donor cell from the PVP-cell suspension and aspirate it in-and-out until the outer membrane has broken down (fragments of the membrane and cytosol should be visible). If necessary a piezo-pulse can be applied. Once a nucleus has been isolated, move it a little bit up in the needle, and continue to pick up nuclei until you have 10-30.</li>
 <li>Move to the drop containing the aligned enucleated oocytes. Fix one oocyte to the holding needle by applying vacuum. Place the nuclear transfer needle (the nuclei should be away from the opening) adjacent to the zona pellucida and apply piezo-pulse until the needle has gone through the zona pellucida. Carefully move one nucleus to the tip of the needle, push the needle into the oocyte until the tip of the needle is 2/3 inside. Apply a small negative pressure, so that a little bit of the oocyte membrane is in the needle.</li>
 <li>The next step is very critical, since it is the only time when the oocyte is actually "open". Apply a single pulse of piezo, push the nucleus out of the needle by applying positive pressure, carefully pull back the needle so that the tip is about 1/3 in the oocyte, and then apply negative pressure and continue to pull back the needle to seal the oocyte. Repeat this step with each oocyte.</li>
 <li>After all oocytes of a group have received a nucleus, they are washed and cultured in KSOM-AA media. Repeat this procedure with all oocytes.</li>
 <li>After the last group has been cultures in KSOM-AA for a minimum of 30 min, the SCNT-embryos are transferred into drops of Ca-free activation media containing SrCl2.</li>
 <li>After 6 hours of activation, the amount of pseudo-pronuclei (PPN)-positive SCNT-embryos is determined. The embryos are then washed and cultured in KSOM-AA drops for additional 3.5 days until they have reached the blastocyst stage.</li>
 <li>Drugs or chemicals of choice (such as Trichostatin A) can be added to the activation and culture media.</li>
 </ol>
 </li>
</ol>
3.Derivation of embryonic stem cells
The SCNT-blastocysts can be used to either transfer them into pseudo-pregnant females (also known as direct or one-step cloning) or to derive embryonic stem cells (also known as indirect or two-step cloning). Since it is much more efficient to generate ES cells, we choose the two-step procedure. In case the scientist is interested in direct cloning, the blastocysts can be transferred directly into pseudo-pregnant females as described in section 5.
There are many protocols describing the derivation of embryonic stem cells. The one described here is a standard technique, which utilizes feeder cells, and fetal calf serum.
<ol>
 <li>The second day after nuclear transfer, prepare a 96-U-well plate for ES cell derivation.</li>
 <li>Add 100 &mu;L Gelatin into each well of a 96-U-well plate, incubate for a minimum of 10 min, and remove it afterwards.</li>
 <li>Thaw feeder cells and resuspend in an according volume of ESD medium, e.g. feeder cells equivalent to an area of 25 cm2 are resuspended in 10 mL ESD medium and 200 &mu;L of the cell suspension is added into each of the gelatin-treated well.</li>
 <li>Put the plate into an incubator and culture at 37&deg;C, 5% CO2.</li>
 <li>After 3.5 days, the embryos should be at the blastocyst stage.</li>
 <li>Prepare a sterile bacterial dish by plating a few drops ES cell medium and acidic thyrode.</li>
 <li>The blastocysts are first transferred from the KSOM-AA drops into drops containing normal ES cell medium and washed twice.</li>
 <li>Carefully transfer a group of blastocysts (5-10 blastocysts) into one drop of acidic thyrode and keep in there until the zona pellucida has dissolved.</li>
 <li>The blastocysts are then transferred into a drop with ES medium, washed twice, and then placed into the feeder-coated 96-U-well plate (one blastocyst into one well).</li>
 <li>The embryos are then cultured for 5-7 days in an incubator at 37&deg;C, 5% CO2 without disturbing them. This gives the embryo time to attach to the feeder layer, and to form an outgrowth.</li>
 <li>Prepare 24-well plates, by plating feeders in ESD medium into each well, e.g. resuspend an equivalent of 50 cm2 feeder in 12 mL ESD medium, and add 500 &mu;L of the cell suspension into each well.</li>
 <li>Carefully remove the ESD medium from the 96-U-well plate with the embryos, wash each well twice with 250 &mu;L Hepes (or PBS), add 50 &mu;L Trypsin and incubate for about 5 min at 37&deg;C.</li>
 <li>Pipette up and down multiple times, until outgrowth has fallen apart into a single-cell suspension, transfer into a 24-well plate and incubate at 37&deg;C, 5% CO2.</li>
 <li>If ESC derivation was successful, ESC colonies should be visible after 7-10 days.</li>
</ol>
4.Blastocyst injection
The injection of blastocysts is a routine technique to generate any kind of transgenic mouse model. It is important to mention that the injection of blastocysts and the subsequent transfer into pseudo-pregnant females needs to be timed well.
<ol>
 <li>Prime female mice with PMS and HCG as described in section 2.1. After HCG injection put female mice together with male mice (one female and one male mouse per cage).</li>
 <li>The next day, check females for plugs. This is counted as 0.5dpc (days post coitus).</li>
 <li>Fertilized embryos are then isolated from the oviduct as described in section 2.1, and cultured in KSOM-AA for additional 3 days.</li>
 <li>Prepare ES cells on the day of blastocyst injection (3.5dpc).</li>
 <li>Remove medium from ES cells, wash twice with Hepes (or PBS), add Trypsin and incubate for 5 min at 37&deg;C.</li>
 <li>Resuspend trypsinized cells with ES medium, plate in a dish, and incubate for 45-60 min at 37&deg;C, 5% CO2 to reduce the amount of feeder cells (feeder cells adhere faster than ES cells).</li>
 <li>Transfer the cell suspension through a cell strainer (40 or 70 &mu;m) into a tube, and centrifuge for 5 min at 1000rpm.</li>
 <li>Remove supernatant, resuspend cell pellet in 500 &mu;L ES medium and store on ice until needed.</li>
 <li>Prepare the lid of a sterile bacterial dish by plating drops containing PVP and HCZB.</li>
 <li>Prepare a 15 &mu;m needle by loading with mercury, placing it in position and washing it with PVP.</li>
 <li>Place a group of blastocysts (10-30 blastocysts) into a drop with HCZB, and add 5-50 &mu;L ES cell suspension (depends on the concentration) into another drop of HCZB.</li>
 <li>Pick up ES cells (50-100 cells) with the injection needle.</li>
 <li>Move to the drop with the blastocysts. Align the blastocysts horizontally, and fix one with the holding needle by applying vacuum.</li>
 <li>Using the injection needle turn the blastocyst until the inner cell mass (ICM) is at the 9 o'clock position.</li>
 <li>Place your injection needle at 3 o'clock, make sure that there are no ES cells at the tip of the needle, and apply piezo-pulse until the injection needle penetrates through the zona pellucida and the trophectoderm into the blastocoel.</li>
 <li>Release a few ES cells (5-15 cells), so that the ES cells stick to the ICM. Continue until all blastocysts of a group have been injected with ES cells.</li>
 <li>After one group of blastocysts is finished, wash them twice in KSOM-AA and keep them in a drop with KSOM-AA until all blastocysts are injected.</li>
</ol>
5. Embryo Transfer
The transfer of embryos into pseudo-pregnant females represents a surgical procedure, which needs to be carried out very carefully and according to the guidelines of the researcher's institute.
<ol>
 <li>Anaesthetize female recipient mice, shave lower right quadrant, and disinfect.</li>
 <li>Using scissors open the skin, and separate the peritoneum from the skin.</li>
 <li>Locate the ovary through the peritoneum (red spot), and open the peritoneum in close proximity to the ovary.</li>
 <li>Using a forceps, carefully grab the oviduct and pull out the uterus.</li>
 <li>Pick up a group of about 10 blastocysts with a capillary connected to a mouth pipette.</li>
 <li>Fix the uterus with one forceps, punch a small whole into it with a small needle, and insert the capillary with the blastocysts into the lumen of the uterus.</li>
 <li>Carefully release the embryos into the uterus, and remove your capillary.</li>
 <li>Push the uterus back into the abdominal cavity.</li>
 <li>Locate the edges of the peritoneum and close it with a few stitches.</li>
 <li>Close the skin of the mouse with a few staples.</li>
 <li>For post-operative pain, administer 5 &mu;g Carprofen per mg of body weight.</li>
</ol>
<img src="/files/ftp_upload/2168/2168fig1.jpg" alt="Figure 1" /> Figure 1. Somatic cell nuclear transfer of pre-defined T cells in B6CF1 background. Absolute and relative numbers of embryos generated from CD8+ T cells and embryonic stem cells derived from SCNT blastocysts.
<img src="/files/ftp_upload/2168/2168fig2.jpg" alt="Figure 2" /> Figure 2. Representative flow cytometric analysis of chimeric mice injected with SCNT embryonic stem cells. Gate and number (percent per total CD8+ T cells) indicates the presence of specific CD8+ T cells in chimeric mice.

<ol>
	<li>Schatz, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V(D)J+recombination.">V(D)J recombination.</a> Immunol. Rev. 200, 5-5 (2004).</li><li>Barnden, M. J., Allison, J., Heath, W. R., Carbone, F. R., R, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+TCR+expression+in+transgenic+mice+constructed+using+cDNA-based+alpha-+and+beta-chain+genes+under+the+control+of+heterologous+regulatory+elements.">Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-chain genes under the control of heterologous regulatory elements.</a> Immunol. Cell Biol. 76, 34-34 (1998).</li><li>Eggan, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hybrid+vigor,+fetal+overgrowth,+and+viability+of+mice+derived+by+nuclear+cloning+and+tetraploid+embryo+complementation.">Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation.</a> Proc Natl Acad Sci U S A. 98, 6209-6209 (2001).</li><li>Kishigami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Significant+improvement+of+mouse+cloning+technique+by+treatment+with+trichostatin+A+after+somatic+nuclear+transfer.">Significant improvement of mouse cloning technique by treatment with trichostatin A after somatic nuclear transfer.</a> Biochem Biophys Res Commun. 340, 183-183 (2006).</li><li>Wakayama, T., Yanagimachi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+cloning+with+nucleus+donor+cells+of+different+age+and+type.">Mouse cloning with nucleus donor cells of different age and type.</a> Mol Reprod Dev. 58, 376-376 (2001).</li></ol>

A patent has been filed.

We have shown here that SCNT can be used to generate transnuclear mice from T cells with pre-defined specificity. Although not shown here, the technique should also be usable to generate transnuclear mice from B cells with pre-defined specificity.
One important thing to consider is the strain and gender of mouse, which is infected or immunized and used to isolate T or B cells of interest. Since T and B cells have a very low reprogramming efficiency in general, we recommend using F1 hybrids of desired haplotype. Because the donor cell also determines the sex, we recommend using T or B cells from male mice. This means that only male chimeric mice would transmit the TCR or BCR through the germline, with male mice being easier and faster to breed. Taken together, we think that epigenetic reprogramming via SCNT represents a powerful tool to generate a novel type of mouse models, which will be of great advantage for the immunological field.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Red Blood Cell lysis Buffer</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>R7757</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Falcon</td>
<td>REF 352340</td>
<td>&nbsp;</td>
<tr>
<td>Pregnant Mare Serum (PMS)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>367222</td>
<td>&nbsp;</td>
<tr>
<td>Human Chorionic Gonadotropin (HCG)</td>
<td>&nbsp;</td>
<td>Calbiochem</td>
<td>230734</td>
<td>&nbsp;</td>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H4272</td>
<td>Type IV-S</td>
</tr>
<tr>
<td>KSOM-AA</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MR-106-D</td>
<td>&nbsp;</td>
<tr>
<td>HCZB</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MR-173-D</td>
<td>Customized medium</td>
</tr>
<tr>
<td>Ca-free medium for activation</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MR-174-D</td>
<td>Customized medium</td>
</tr>
<tr>
<td>SrCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>255521</td>
<td>100mM = 10x</td>
</tr>
<tr>
<td>AlbuMAX I</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11020-021</td>
<td>&nbsp;</td>
<tr>
<td>PVP</td>
<td>&nbsp;</td>
<td>MP Biotech</td>
<td>102787</td>
<td>Make 10% solution using HCZB, and add 0.1% AlbuMAX I</td>
</tr>
<tr>
<td>Cytochalasin B</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C6762</td>
<td>500&mu;g/ml = 100x</td>
</tr>
<tr>
<td>Trichostatin A</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T8552</td>
<td>5mM = 1000x</td>
</tr>
<tr>
<td>Mineral Oil</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M5310</td>
<td>&nbsp;</td>
<tr>
<td>Holding needle</td>
<td>&nbsp;</td>
<td>Humagen</td>
<td>MPH-SM-30</td>
<td>&nbsp;</td>
<tr>
<td>Injection and transfer needles</td>
<td>&nbsp;</td>
<td>Humagen</td>
<td>4-15, 7-15, 15-15</td>
<td>&nbsp;</td>
<tr>
<td>Mouth pipette</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5177</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Instruments</td>
<td>14088-10</td>
<td>or something similar</td>
</tr>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Instruments</td>
<td>11251-10</td>
<td>or something similar</td>
</tr>
<tr>
<td>Wound Clip Applicator</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427630</td>
<td>&nbsp;</td>
<tr>
<td>Wound Clips</td>
<td>&nbsp;</td>
<td>BD</td>
<td>427630</td>
<td>&nbsp;</td>
<tr>
<td>Suture</td>
<td>&nbsp;</td>
<td>Ethicon</td>
<td>K871H</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>D6429</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>SH30071.03</td>
<td>15%</td>
</tr>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>17-602E</td>
<td>100x</td>
</tr>
<tr>
<td>Glutamin</td>
<td>&nbsp;</td>
<td>MP Biomedicals</td>
<td>101806</td>
<td>200mM = 100x</td>
</tr>
<tr>
<td>Non-essential Aminoacids</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11140050</td>
<td>100x</td>
</tr>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985-023</td>
<td>5&mu;l in 500ml</td>
</tr>
<tr>
<td>LIF</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ESG1106</td>
<td>1000x</td>
</tr>
<tr>
<td>MEK inhibitor</td>
<td>&nbsp;</td>
<td>Cell Signaling</td>
<td>9900L</td>
<td>For ES derivation only, add to ES medium (final concentration 50&mu;M)</td>
</tr>		</tbody>
		</table>
		</div>

transnuclear mice with pre-defined t cell receptor specificities against toxoplasma gondii obtained via scnt

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Watch this Scientific Journal Video about Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT at JoVE.com

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

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

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

transnuclear-mice-with-pre-defined-t-cell-receptor-specificities

Research

JoVE Journal

Immunology and Infection

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with causing food- and water-borne disease outbreaks. The definitive host is the feline species where sexual replication occurs resulting in the development of the highly infectious and environmentally resistant oocyst. Infection occurs via ingestion of tissue cysts from contaminated meat or oocysts from soil or water. Infection is typically asymptomatic in healthy individuals, but results in a life-long latent infection that can reactivate causing toxoplasmic encephalitis and death if the individual becomes immunocompromised. Meat contaminated with T. gondii cysts have been the primary source of infection in Europe and the United States, but recent changes in animal management and husbandry practices and improved food handling and processing procedures have significantly reduced the prevalence of T. gondii cysts in meat1, 2. Nonetheless, seroprevalence in humans remains relatively high suggesting that exposure from oocyst contaminated soil or water is likely. Indeed, waterborne outbreaks of toxoplasmosis have been reported worldwide supporting the theory exposure to the environmental oocyst form poses a significant health risk3-5. To date, research on understanding the prevalence of T. gondii oocysts in the water and environment are limited due to the lack of tools to detect oocysts in the environment 5, 6. This is primarily due to the lack of efficient purification protocols for obtaining large numbers of highly purified T gondii oocysts from infected cats for research purposes. This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation7.

Obtaining Highly Purified Toxoplasma gondii Oocysts by a Discontinuous Cesium Chloride Gradient

This study describes the development of a modified CsCl method that easily purifies T. gondii oocysts from feces of infected cats that are suitable for molecular biological and tissue culture manipulation

Toxoplasma gondii is an obligate intracellular protozoan pathogen that commonly infects humans. It is a well characterized apicomplexan associated with ...

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii disseminates as a fast replicating form called the tachyzoite. Tachyzoites convert into a slow-growing encysted form called the bradyzoite by a signaling process that is not well characterized. Within animals, bradyzoite cysts are found in the central nervous system and muscle tissue and represent the chronic stage of infection. Conversion to bradyzoites can be simulated in tissue culture by CO2 starvation, using medium with high a pH, or the addition of interferon gamma (IFN&gamma;). Bradyzoites are characterized by the presence of a cyst wall, to which the lectin Dolichos biflorus agglutinin (DBA) binds. Fluorescently labeled DBA is used to visualize the cyst wall in parasites grown in human foreskin fibroblasts (HFFs) that have been exposed to low CO2 and high pH medium. Similarly, parasites residing in murine bone marrow-derived macrophages (BMMs) display a cyst wall detectable by DBA after the BMMs are activated with IFN&gamma; and lipopolysaccharide (LPS). This protocol will demonstrate how to induce conversion of T. gondii to bradyzoites using a high pH growth medium with low CO2 and activation of BMMs. Host cells will be cultured on coverslips, infected with tachyzoites and either activated with addition of IFN&gamma; and LPS (BMMs) or exposed to a high pH growth medium (HFFs) for three days. Upon completion of infections, host cells will be fixed, permeabilized, and blocked. Cyst walls will be visualized using rhodamine DBA with fluorescence microscopy.

Toxoplasma gondii Cyst Wall Formation in Activated Bone Marrow-derived Macrophages and Bradyzoite Conditions

Toxoplasma gondii converts to a cyst form in response to environmental stresses, which can be mimicked in tissue culture models. This video demonstrates techniques to examine cyst wall formation by activating bone marrow-derived macrophages or changing growth medium pH in fibroblast cells.

Toxoplasma gondii is an obligate intracellular parasite that can invade any nucleated cell of warm-blooded animals. During infection, T. gondii ...

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm-/- and p53-/- consistently develop thymic lymphoma early in life 1,2, and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols 1,3. Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain 1,4. We have successfully established several T-cell lines using this protocol and inbred strains ofAtm-/- [FVB/N-Atmtm1Led/J] 2 and p53-/- [129/S6-Trp53tm1Tyj/J] 5 mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.

Derivation of Thymic Lymphoma T-cell Lines from Atm-/- and p53-/- Mice

In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm-/- and p53-/- mice with thymic lymphoma.

Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified ...

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth defects upon congenital infection. The lytic replication cycle is characterized by three stages: 1. active invasion of a nucleated host cell; 2. replication inside the host cell; 3. active egress from the host cell. The mechanism of egress is increasingly being appreciated as a unique, highly regulated process, which is still poorly understood at the molecular level. The signaling pathways underlying egress have been characterized through the use of pharmacological agents acting on different aspects of the pathways1-5. As such, several independent triggers of egress have been identified which all converge on the release of intracellular Ca2+, a signal that is also critical for host cell invasion6-8. This insight informed a candidate gene approach which led to the identification of plant like calcium dependent protein kinase (CDPK) involved in egress9. In addition, several recent breakthroughs in understanding egress have been made using (chemical) genetic approaches10-12. To combine the wealth of pharmacological information with the increasing genetic accessibility of Toxoplasma we recently established a screen permitting the enrichment for parasite mutants with a defect in host cell egress13. Although chemical mutagenesis using N-ethyl-N-nitrosourea (ENU) or ethyl methanesulfonate (EMS) has been used for decades in the study of Toxoplasma biology11,14,15, only recently has genetic mapping of mutations underlying the phenotypes become routine16-18. Furthermore, by generating temperature-sensitive mutants, essential processes can be dissected and the underlying genes directly identified. These mutants behave as wild-type under the permissive temperature (35 &deg;C), but fail to proliferate at the restrictive temperature (40 &deg;C) as a result of the mutation in question. Here we illustrate a new phenotypic screening method to isolate mutants with a temperature-sensitive egress phenotype13. The challenge for egress screens is to separate egressed from non-egressed parasites, which is complicated by fast re-invasion and general stickiness of the parasites to host cells. A previously established egress screen was based on a cumbersome series of biotinylation steps to separate intracellular from extracellular parasites11. This method also did not generate conditional mutants resulting in weak phenotypes. The method described here overcomes the strong attachment of egressing parasites by including a glycan competitor, dextran sulfate (DS), that prevents parasites from sticking to the host cell19. Moreover, extracellular parasites are specifically killed off by pyrrolidine dithiocarbamate (PDTC), which leaves intracellular parasites unharmed20. Therefore, with a new phenotypic screen to specifically isolate parasite mutants with defects in induced egress, the power of genetics can now be fully deployed to unravel the molecular mechanisms underlying host cell egress.

A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants

Forward genetics is a powerful method to unravel the molecular level of how Toxoplasma egresses from its host cell. Protocols are provided to chemically mutagenize parasites, enrich for mutants with defects in induced egress, and validate the phenotype of cloned mutants.

The widespread, obligate intracellular, protozoan parasite Toxoplasma gondii causes opportunistic disease in immuno-compromised patients and causes birth ...

Genetic Manipulation in &Delta;ku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii by deleting the gene encoding the KU80 protein1,2. The &Delta;ku80 strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro and in vivo and exhibit essentially a 100% frequency of homologous recombination. The &Delta;ku80 strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4.
Here, we report methods for using type I and type II &Delta;ku80&Delta;hxgprt strains to advance gene targeting approaches in T. gondii. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT) selectable marker. The described gene targeting protocol can be used in a variety of ways in &Delta;ku80 strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii and related significant human pathogens that cause malaria (Plasmodium sp.) and cryptosporidiosis (Cryptosporidium).

Here we report a method for using type I and type II &Delta;ku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene ...

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within virtually any warm blooded vertebrate cell type. During parasite invasion of a host cell, the parasite creates a parasitophorous vacuole (PV) that originates from the host cell membrane independent of phagocytosis within which the parasite replicates. While IFN-dependent-innate and cell mediated immunity is important for eventual control of infection, innate immune cells, including neutrophils, monocytes and dendritic cells, can also serve as vehicles for systemic dissemination of the parasite early in infection. An approach is described that utilizes the host innate immune response, in this case macrophages, in a forward genetic screen to identify parasite mutants with a fitness defect in infected macrophages following activation but normal invasion and replication in na&#239;ve macrophages. Thus, the screen isolates parasite mutants that have a specific defect in their ability to resist the effects of macrophage activation. The paper describes two broad phenotypes of mutant parasites following activation of infected macrophages: parasite stasis versus parasite degradation, often in amorphous vacuoles. The parasite mutants are then analyzed to identify the responsible parasite genes specifically important for resistance to induced mediators of cell autonomous immunity. The paper presents a general approach for the forward genetics screen that, in theory, can be modified to target parasite genes important for resistance to specific antimicrobial mediators. It also describes an approach to evaluate the specific macrophage antimicrobial mediators to which the parasite mutant is susceptible. Activation of infected macrophages can also promote parasite differentiation from the tachyzoite to bradyzoite stage that maintains chronic infection. Therefore, methodology is presented to evaluate the importance of the identified parasite gene to establishment of chronic infection.

Forward Genetics Screens Using Macrophages to Identify Toxoplasma gondii Genes Important for Resistance to IFN-&#947;-Dependent Cell Autonomous Immunity

Forward genetics is a powerful approach to identify genes in intracellular pathogens important for resistance to cell autonomous immunity. The current approach uses innate immune cells, specifically macrophages, to identify novel Toxoplasma gondii genes important for immune evasion.

Toxoplasma gondii, the causative agent of toxoplasmosis, is an obligate intracellular protozoan pathogen. The parasite invades and replicates within ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...