Using the Xenopus ORFeome

with the pDXTR family of transgenesis vectors

Xenopus PI Meeting

Murielle Chimi
Daniel R. Buchholz
Dep't of Biological Sciences
University of Cincinnati
Cincinnati, OH USA
 pDXTR
vector for transgenic expression of Xenopus ORFs

Talk Outline:
Introduction
pDXTR features
pDXTR validation
Origins of pDXTR
pDXTR
 pDXTR
"plasmid destination Xenopus transgenesis red"

pDXTR features:
Gateway cloning site for ORFs
MCS for promoter of interest
tissue-specific inducible expression of ORF
ORF expression verification
multiple ORF tag options
transgenesis options and markers

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

 pDXTR Features I

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

Gateway cloning site

ORF expression verification
multiple ORF tag options
 pDXTR Features 2

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

MCSI Tet-On 3G polyA HS4 ext TRE3G

tissue-specific inducible expression of ORF

 pDXTR Features 3

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

MCS IVa
CRY mCherry polyA MCS IVb

transgenesis markers
 pDXTR Features 4
SceI
fC31 tol2 5' HS4 ext FRT
loxP

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

lox2272 FRT3 HS4 ext tol2 3' SceI

transgenesis options
 pDXTR verification

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

Gateway Cloning

Tg MCS rtTA tetO GFP T2 Red-H2B CRY mChe Tg

pDXTR-GFP
Tg(TRE3G:GFP-T2-mCherry-hist2h2be,cryga:mCherry)
 Inject pDXTR-GFP into rtTA transgenic line
pCAR rtTA line
pCAR rtTA Xla.Tg(actc1:rtTA,TRE:DNthra-GFP)

Tg MCS rtTA tetO GFP T2 Red-H2B CRY mChe Tg

pDXTR-GFP
+ Dox
- Dox
 Inject pDXTR-GFP into rtTA transgenic line
pCAR rtTA line
pCAR rtTA Xla.Tg(actc1:rtTA,TRE:dnthra-GFP)

Tg MCS rtTA tetO GFP T2 Red-H2B CRY mChe Tg

pDXTR-GFP
 pDXTR vectors

Tg MCS rtTA tetO Gateway T2 Red-H2B CRY mChe Tg

Tg MCS rtTA tetO ORF T2 Red-H2B CRY mChe Tg

Tg POI rtTA tetO ORF T2 Red-H2B CRY GFP Tg

Tg MCS Gateway T2 Red-H2B CRY mChe Tg

 Summary
pDXTR is a versatile transgenesis vector for Xenopus ORFeome

Key features are:

Gateway cloning of ORFs
tissue-specific, inducible control of ORF expression
multiple options for ORF tags
multiple transgenesis methods and markers
 Acknowledgements

Marcin Wlizla Jinyoung Choi

Aaron Zorn Chris Lawrence

Michael Gilchrist
David Hill
Todd Stukenberg
Aaron Zorn
 rtTA Transgenic Line for testing pDXTR
rtTA line: pCAR rtTA Xla.Tg(actc1:rtTA,TRE:dnthra-GFP)

TRE line: TRE EGFP CRY DsRed Xla.Tg(Ptight:eGFP,crya:DsRed)

Double Transgenic
+ Dox, 1day
+ Dox, 1day

non-Tg rtTA line TRE line