Name: retroviral transduction of bone marrow progenitor cells to generate t-cell receptor retrogenic mice
Uploaded: 2016-07-11T15:00:00
Description: Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

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.

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	<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 ...

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