Introducing a mouse (VeDTR system) that allows
you to specify two marker genes and remove onlycells expressing both.
Accurate research by deleting “cells” rather than deleting “genes” that have wideranging effects

Masahiro Yamamoto
Laboratory of Immunoparasitology, IFReC, Osaka University
Department of Immunoparasitology, RIMD, Osaka University
CIDER, Osaka University

CD4 T cells have recently been differentiated into many more subsets.
How can we delete and analyze "only" subdivided cells (e.g. Th1-Treg)?
Naïve CD4+T cell (Th0)CD4+Tcell

Th1

Th2Treg

Th17TfhTbet

Gata3

Foxp3

Rorγt

Bcl6

Th1-Treg

Th2-TregTh17-Treg

Tfh-Treg

Foxp3

Foxp3

Tbet

Gata3

Foxp3
Rorgt

Foxp3
Bcl6

Treg cell subsets（Subset of CD4+ T cellsubset）

Tbet

Tbet KO×Foxp3 KO＝Double KO？
→Many cells are collaterally deleted

Foxp3

Here!
(Th1-Treg)

Tbet KO
Foxp3

Many cells expressing only Tbet and Foxp3 are also involved
2Interest is only cells that express both Foxp3 and Tbet

Using Cre-loxP and Flp-FRT, only cells expressing both emit
fluorescence or can be removed with drugs (VeDTR system) Tbet
Treg

全部

Foxp3

Th1

全部

Deletedonly here

VeDTR mouse×

Promoter X- Cre×

Promoter Y- Flp=Mice where only thetargeted cells emit
fluorescence and are
killed byadministration ofdiphtheria toxin

Development and growth are the same as normal mice, target cells can beanalyzed using fluorescence, and only target cells can be removed byadministering diphtheria toxin.
Patent Pending（PCT/JP2023/025274）

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Cell-specific KO is possible in any cell fraction if two sets ofcharacteristic genes that define that cell are found.
Gene X

Various immune cells

Gene Y<Models>
InfectionTumor

X and Y

DiseaseGene X Gene Y

Currently, it is difficult to elucidate thefunction of the cell subset of interest

Novel
VeDTR mouse system

Gene X-Cre
Gene Y-Flp
VeDTR mouse
Targeted labeling and disruptionof thecell subset
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More detailed analysis of target cells is possible using fluorescenceas an indicator.
spleenFoxp3-Cre / Tbx21-Flp
VeDTR micevs
YFPα-CD3/CD28

125vs
YFP+401003

Sorting ofCD4+ CD25+T cells

Isolation of
YFP+ or YFPTreg

Prf1

perforin 1 (pore forming protein)

Mt3

metallothionein 3

Ctla2bcytotoxic T lymphocyte-associated protein 2 beta

Cd68

CD68 antigen

Lat2"linker for activation of T cells family, member 2"

Spp1

secreted phosphoprotein 1 （Osteopontin）

Ifng

interferon gamma

Srxn1

sulfiredoxin 1 homolog (S. cerevisiae)

Havcr2

hepatitis A virus cellular receptor 2

Prdm1"PR domain containing 1, with ZNF domain"

Ccl3

chemokine (C-C motif) ligand 3

Il10interleukin 10

Adam8a disintegrin and metallopeptidase domain 8

Ccr5chemokine (C-C motif) receptor 5

Ccl4

chemokine (C-C motif) ligand 4

Ttc39ctetratricopeptide repeat domain 39C

Tnfrsf8"tumor necrosis factor receptor superfamily, member 8"

Gzmb

granzyme B

Mt2

metallothionein 2

Nfil3"nuclear factor, interleukin 3, regulated"Tigit

T cell immunoreceptor with Ig and ITIM domains

Pcyt1a"phosphate cytidylyltransferase 1, choline, alpha isoform"

Mt1

metallothionein 1

Tubb6"tubulin, beta 6 class V"

Il1r2"interleukin 1 receptor, type II"

Adap1ArfGAP with dual PH domains 1

Nkg7

natural killer cell group 7 sequence

Dgat1

diacylglycerol O-acyltransferase 1

Ptpn5"protein tyrosine phosphatase, non-receptor type 5"

α-CD3/α-CD28
刺激

Bulk RNAseq

It is also possible to search forcharacteristic markers of cells gatheredin diseased areas.

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It is also possible to remove the target cells by administering DT andobserve the effects.

Foxp3-Cre / Tbx21-Flp
VeDTR mice

B16F10 melanoma
Subcutaneous implant

AB

CD4

CD4+T

YFP+ / CD4+

4030

1.94

**1200800400%

DTPBS (n=8)
DT (n=9)

1600

✱✱✱

5035.3

B16F102000mm3 (+SEM)

PBSFACSandmeasurement ofTumor volumePBSorDT treatment
from Day 10

20010YFP

101112131415
Days post tumor inoculation

160PBSDT

Removal of Th1-Treg leads to tumor (B12melanoma) growth
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looking for technology license and new joint research.

VeDTR mouse・Analysis of specific subsets of subdivided immune cells is
possible with fluorescence and cell deletion
・Can be removed at any time without affecting growth anddevelopment
・Target cells are not limited to the immune system
・Can also be used to create new disease model mice
Please tell us your wishes

Cell Reports Volume 42, Issue 7, 25 July 2023, 112813
doi: 10.1016/j.celrep.2023.112813. Epub 2023 Jul 12.

A genetic method specifically delineates Th1-type Treg cells andtheir roles
in tumor immunity
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