This work was supported by grants from NIH (5K22A1119151-01 and 1R56DK104903-01) to M.L.B, Pilot/Feasibility Program of the Diabetes Research Center (P30-DK079638) at BCM, JDRF 1-FAC-2014-243-A-N APF, ADA 1-15-JF-07, AAI Careers in Immunology Fellowship to M.B., and The Robert and Janice McNair Foundation.

Ethics Statement: Every effort is made to keep animal discomfort or stress to a minimum during irradiation and tail vein injections. Mice are used as a source of cells in these experiments; as such there are no procedures or manipulations apart from euthanasia. Mice will be euthanized by CO2 inhalation followed by cervical dislocation to confirm death. This procedure is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.
1. Prepar...

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In the protocol, we detail several critical steps to ensure optimal bone marrow health, transduction efficiency and reconstitution. First critical step is the generation and proper maintenance of the GP+E86 viral producer cells. Use early passage producer cell lines and maintain at 80% confluency or lower prior to use. When making fresh GP+E86 viral producer cells, ensure the 293T cells are early passage and growing in culture for 24 - 48 hr. Plating too many GP+E86 cells during the transduction step will lower viral tit...

T cell receptor (TCR) repertoire of humans and mice has been estimated at 1 x 108 and 2 x 106 unique TCRs respectively1,2. This large diversity allows T cells to recognize a vast array of antigen epitopes derived from self-peptides as well as from pathogens presented by the major histocompatibility complex (MHC) on antigen presenting cells (APCs). The subtle differences in the interactions of the TCRs with unique peptide-MHC complexes dictate whether a T cell will undergo apoptosis, anergy, activation, differentiation, cytokine production or cytotoxicity. However, due to the large TCR repertoire, analysis of how a specific TCR will respond to a particular antigen requires the use of single TCR systems.
Various TCR transgenic mice have been generated in order to study the function of a single TCR in an in vivo model 3-9. However, there are caveats to TCR transgenic mice including the cost, the length of time to generate a single transgenic mouse and the so called founder effect of random transgene insertion into germline DNA10. Therefore, relatively few TCR transgenic mice have been generated for any given antigen and the functional implications of high and low TCR affinity for the same epitope are rarely addressed. To address the need for a rapid approach to screen and study multiple TCRs individually or in combination, retrogenic (&#39;retro&#39; from retrovirus and &#39;genic&#39; from transgenic) mice have been utilized as an alternative to TCR transgenic mice11-13.
The 2A peptide consensus motif found within several viruses consist of an 2A-Asp-Val/Ile-Glut-X-Asn-Pro-Gly-2B-Pro, in which cleavage occurs between the glycine of the 2A and the proline of the 2B from cis-acting hydrolase activity, resulting in ribosomal skipping during translation10,14-16. For a detailed diagram depicting the cleavage of the various 2A peptides (F2A, E2A, T2A and P2A) please refer to references 10 - 12. In this manner, 2 cistrons (TCR alpha and TCR beta) can be linked resulting in stoichiometric translation in a single vector. Utilizing this approach, we are able to express and directly compare multiple antigen specific TCRs in vivo.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM, high glucose + glutamine</td>
			<td>Corning Cellgro</td>
			<td>10-013-CV</td>
			<td>Dulbecco&#39;s Modification of Eagle&#39;s Medium with 4.5 g/L glucose, L-glutamine &#38; sodium pyruvate</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biological</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-Versene</td>
			<td>Lonza</td>
			<td>17-161F</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 um syringe filter</td>
			<td>Thermo Scientific</td>
			<td>194-2545</td>
			<td></td>
		</tr>
		<tr>
			<td>polybrene</td>
			<td>Sigma</td>
			<td>H9268-10G</td>
			<td>Sterile Filtered in dH2O</td>
		</tr>
		<tr>
			<td>Ciprofloxacin&#160;</td>
			<td>VWR</td>
			<td>AAJ61970-06</td>
			<td></td>
		</tr>
		<tr>
			<td>5-fluorouracil (5-FU)</td>
			<td>VWR</td>
			<td>AAA13456-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Corning Cellgro</td>
			<td>25-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM nonessential Amino Acids</td>
			<td>Corning Cellgro</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M solution</td>
			<td>Corning Cellgro</td>
			<td>25-060-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Gibco by Life Technologies</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen/Strept</td>
			<td>Corning Cellgro</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Corning Cellgro</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>150 mm tissue culture dishes</td>
			<td>Greiner Bio-one</td>
			<td>639160</td>
			<td></td>
		</tr>
		<tr>
			<td>Tisue culture-treated 6-well flat plate</td>
			<td>Greiner Bio-one</td>
			<td>657160</td>
			<td></td>
		</tr>
		<tr>
			<td>70 um nylon cell strainers</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IL-3</td>
			<td>Invitrogen</td>
			<td>PMC0033</td>
			<td></td>
		</tr>
		<tr>
			<td>Human IL-6</td>
			<td>Invitrogen</td>
			<td>&#160;PHC0063</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Stem Cell Factor</td>
			<td>Invitrogen</td>
			<td>PMC2113L</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Corning Cellgro</td>
			<td>46-D13-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>HANKS Buffer</td>
			<td>Corning Cellgro</td>
			<td>21020147</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 10 mL Syringe</td>
			<td>BD</td>
			<td>300912</td>
			<td></td>
		</tr>
		<tr>
			<td>BD 1 mL Syringe</td>
			<td>BD</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr>
			<td>27G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305109</td>
			<td></td>
		</tr>
		<tr>
			<td>30G x 1/2 BD Precision Glide Needle</td>
			<td>BD</td>
			<td>305106</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Bone marrow transduction efficiency is checked at step 13.3 of the protocol before the bone marrow is injected into tail vein i.v. In the representative bone marrow transduction figure (Figure 1A), approximately 10-&#956;l of the harvested bone marrow was added to 100-&#956;l of PBS and analyzed for ametrine expression. Generally the percentage of fluorescent positive cells is between 25% and 70%, depending on the construct and retroviral titer. After 6 weeks bone marrow ...

Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

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

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 ...

The goal of this procedure is to generate retrogenic mice to determine the in vivo function of multiple t-cell receptors. This is accomplished by utilizing a retroviral construct that encodes the alpha and beta chains of the t-cell receptor, linked by a 2A peptide consensus motif. This method can actually help answer key questions in the immunology field, by screening multiple TCRs specific for multiple antigens, you can actually determine their in vivo function.The main advantage of this technique is that once the retroviral construct is completed, the t-cell compartment of the retrogenic mice will be reconstituted approximately around seven weeks. Additionally, each retrogenic mouse is essentially a founder, so by analyzing multiple mice, the founder effect is eliminated. Demonstrating this part of the procedure will be Dr.Maria Bettini.Set up for the bone marrow extraction by placing sterile dissection scissors and forceps in the surgical area and by chilling dissection medium in a 50 milliliter conical tube on ice. After euthanizing the mouse and confirming death by cervical dislocation, use dissection scissors to cut up and under the pelvic girdle to remove it. Then cut back down along the femur to the tibia and carefully remove the femur, tibia and humerus.Transfer the bones into a dish of dissection medium in the tissue culture hood and use scissors and forceps to remove all surrounding tissue to obtain clean bones. Separate the bones by cutting at the joints, making sure to cut both ends off each bone. Then insert a 25 gauge needle attached to a 10 milliliter syringe containing dissection medium into a bone.Apply pressure and flush all bone marrow through a 70 micron strainer, resting in a 50 milliliter conical tube. Periodically, push the bone marrow through the 70 micron filter using the plunger of a one milliliter syringe and rinse the strainer with medium. Centrifuge the bone marrow single cell suspension at 300 times g for 10 minutes at four degrees celsius.Following the centrifugation, decant or aspirate the medium and resuspend each cell pellet in one milliliter of ammonium chloride based red blood cell lysis buffer. Incubate at room temperature for one minute. Next, quench the red cell lysis buffer by adding 10 milliliters of plating medium and then centrifuge as before.After removing the supernatant, resuspend the bone marrow in five milliliters of plating medium and perform a cell count using neubauer counting chamber. The yield should be approximately five to 10 times 10 to the sixth cells per mouse, but this can vary between strains. Add 30 milliliters of plating medium, supplemented with freshly thawed IL-3, IL-6 and SCF to 150 millimeter tissue culture plates.Then, add four times 10 to the seventh bone marrow cells to each plate and incubate over night at 37 degrees celsius with five percent carbon dioxide. Demonstrating this part of the procedure will be Thomas Lee. Next, plate three times 10 to the sixth retroviral producer cells in 150 millimeter plates containing 18 milliliters of plating medium.Incubate the retroviral producer cells overnight. The next day, use a 10 milliliter syringe and 0.45 micron filter to harvest and filter the retroviral supernatant from the producer plates. Carefully replace the removed retroviral supernatant with 18 milliliters of fresh plating medium.Supplement the filtered retroviral supernatant with IL-3, IL-6, MSCF and hexadimethrine bromide. Next, move on to harvest the bone marrow cells by first transferring the medium containing non-adherent cells into a sterile 50 milliliter tube. Then wash the plate vigorously with 10 milliliters of PBS and add this to the 50 milliliter conical tube.After centrifuging the cells at 300 times g for 10 minutes at four degrees celsius, decant the supernatant and then resuspend the cells in one to three milliliters of plating medium. After performing a cell count, place bone marrow cells into the soup. Plate this cell mixture at a volume of three milliliters per well in a six well tissue culture plate.Wrap the plates containing bone marrow cells in plastic wrap to minimize gas exchange and then spin the plates at 1, 000 times g for 60 minutes at 37 degrees celsius. After the spin in complete, remove the plastic wrap and place the plates in a 37 degree celsius carbon dioxide incubator for 24 hours. After 24 hours, collect fresh viral supernatant from the viral producer cell line.At this point, the cells should be well spaced with a defiant morphology. Next, carefully aspirate the top two milliliters of medium from each well of the six well plate containing the bone marrow cells. Avoid aspirating the bone marrow cells that are usually concentrated in the middle of the well.Add three milliliters of freshly supplemented viral supernatant to each well of the six well plate containing bone marrow cells. After wrapping the plates in plastic, as before, repeat the spin transduction, and then place the unwrapped plates in the tissue culture incubator. After 24 hours, on day three, remove and replace two milliliters of media from the wells.On day four, collect the bone marrow cells from the six well plate by vigorously washing with PBS and then collecting the cell suspension in a 50 milliliter tube. After centrifuging the cells at 300 times g for 10 minutes at four degrees celsius, decant the supernatant and resuspend the bone marrow in one milliliter of PBS plus 0.5 percent heat inactivated FBS. Place the tube containing the cells on ice.Then, after performing a cell count, resuspend the bone marrow in a sufficient volume of PBS plus 0.5 percent heat inactivated FBS, to inject a minimum of two times 10 to the sixth cells in a 200 to 300 microliter volume per mouse. Finally, with a micropipetter, take a 10 microliter sample of each bone marrow condition and add this to 100 microliters of PBS for analysis of transduction efficiency by flow cytometry. Transduction efficiency of bone marrow cells was analyzed based on the fluorescent marker ametrine.The left panel shows the forward and side scatter of bone marrow cells after five days in culture. The right panel is gated as ametry impositive and includes the transduced bone marrow cells. A successful bone marrow transduction will yield a percentage of fluorescent positive cells between 25 and 70 percent.This image shows t-cell receptor retrogenic t-cells in peripheral blood at six weeks post bone marrow reconstitution. After lysis of red blood cells, the remaining cells were stained for t-cell receptor beta. The right panel shows a representative analysis for ametrine expression and anti t-cell receptor beta.After watching this video, you should have a good understanding of to retrovirally transduce and culture bone marrow cells to regenerate retrogenic mice.

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Immunology and Infection

9.4K Views.  Baylor College of Medicine. The goal of this procedure is to generate retrogenic mice to determine the in vivo function of multiple t-cell receptors. This is accomplished by utilizing a retroviral construct that encodes the alpha and beta chains of the t-cell receptor, linked by a 2A peptide consensus motif. This method can actually help answer key questions in the immunology field, by screening multiple TCRs specific for multiple antigens, you can actually determine their in vivo function.The main advantage of t...