The Ctv-a transgene construct

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The Ctv-a transgene construct Powered By Docstoc
					The Ctv-a transgene construct

The Ctv-a transgene construct (pmCNP-tv-a-IRES-LacZ) was generated using the pTG8

plasmid (Karin Forsberg-Nilsson, Uppsala University, Uppsala, Sweden). The 3.9 kb

fragment of mouse Cnp promoter 1 and 2 (Michel Gravel, McGill University, Montreal)

was cloned into the XbaI-HindIII site of pTG8. A 786 bp fragment of tv-a was amplified

from a Gtv-a vector (Eric Holland, Memorial Sloan-Kettering Cancer Center, New York)

using primers adding two base pairs, to get in frame with the ATG of the Cnp promoter 2,

and ClaI sites at each end. Tv-a was cloned into the ClaI site downstream of the Cnp

promoter in pTG8. A 4.4 kb PDGF-B-IRES-lacZ fragment was cut out from pTG8-

PDGF-B-IRES-lacZ, ligated into pGEM-T Easy, the PDGF-B fragment removed with

BamHI, vector religated and IRES-lacZ cut out with SalI. The 3.7 kb SalI IRES-lacZ

fragment was subcloned into the SalI site downstream of the Cnp promoter and tv-a in

pTG8. The 8.5 kb Not1-Sal1 fragment was excised and purified for transgene production.

Modified oligodendrocyte and oligodendrocyte precursor selective SATO media

[13.4 mg/ml D-MEM (Invitrogen, Stockholm, Sweden), 2 mg/ml NaHCO3, 10 μg/ml

transferrin (Sigma-Aldrich, Stockholm, Sweden), 10 µg/ml insulin (Sigma-Aldrich),

0.161 μg/ml putrescine (Sigma-Aldrich), 0.062 μg/ml progesterone (Sigma-Aldrich), 0.34

μg/ml TIT (Serva, Heidelberg, Germany), 5 ng/ml sodium selenite (Serva), 0.403 µg/ml

L-thyroxin (Serva), 50 mg/ml gentamicin (Boehringer Mannheim, Bromma, Sweden) and

1 % heat inactivated horse serum (Gibco, purchased through Invitrogen, Stockholm,


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