Técnica de las 5S

2.5.1 Vectors

• pApuro[113], gift of Dr. Seth Corey, Pitsburgh, USA • pCDNA3.1/Hisc A, Invitrogen, Karlsruhe, Germahy • pCDNA6A, Invitrogen, Karlsruhe, Germahy

• pMSCV-IRES-EGFP, gift of Dr. Van Etten, Tufts-New England Medical Center, Boston, MA, USA

• ecopack,gift of Dr. Van Etten, Tufts-New England Medical Center, Boston, MA, USA

• MIY,gift of Dr. C. Buske, University of Munich, Germany • pMSCV neo,Clontech, Palo Alto, USA

• pCDNA3 GFP, gift of Dr. K. Foster, University of Munich, Ger- many

2.5.2 All other plasmids used for this work

• pApuro Hck wt,gift of Dr. Seth Corey, Pitsburgh, USA

• pCDpuro Hck K269R,gift of Dr. M. Warmuth, University of Mu- nich, Germany

• pApuro Hck Y501F,gift of Dr. M. Warmuth, University of Munich, Germany

• pApuro Lyn, gift of Dr. M. Warmuth, University of Munich, Ger- many

• pApuro Fyn, gift of Dr. M. Warmuth, University of Munich, Ger- many

• pApuro c-Src,gift of Dr. M. Warmuth, University of Munich, Ger- many

• pCDpuro Hck K269R R150L,generated by mutagenesis • pCDpuro Hck K269R W93A ,generated by mutagenesis • pApuro Hck G2A,generated by mutagenesis

• pCDNA3.1/Hisc A Hck wt. To produce this plasmid PCR frag- ment generated using pApuro Hck wt as a template and the following primers: 5’-GAA TGT GAA TTC ATG GGG TGC ATG AAG TCC AAG-3’ and 5’-CGG GGT ACC TGG CTG CTG TTG GTA CTG G-3’, was sub-cloned into EcoRI/KpnI sites of pCDNA3.1/Hisc A vector.

• pCDNA3.1/Hisc A Hck K269R. To produce this plasmid PCR fragment generated using pCDpuro Hck K269R as a template and the following primers: 5’-GAA TGT GAA TTC ATG GGG TGC ATG AAG TCC AAG-3’ and 5’-CGG GGT ACC TGG CTG CTG TTG GTA CTG G-3’, was sub-cloned into EcoRI/KpnI sites of pCDNA3.1/Hisc A vector.

• pCDNA3.1/Hisc A Hck K269R R150L. To produce this plas- mid PCR fragment generated using pCDpuro Hck K269R R150L as a template and the following primers: 5’-GAA TGT GAA TTC ATG GGG TGC ATG AAG TCC AAG-3’ and 5’-CGG GGT ACC TGG CTG CTG TTG GTA CTG G-3’, was sub-cloned into EcoRI/KpnI sites of pCDNA3.1/Hisc A vector.

mid PCR fragment generated using pCDpuro Hck K269R W93A as a template and the following primers: 5’-GAA TGT GAA TTC ATG GGG TGC ATG AAG TCC AAG-3’ and 5’-CGG GGT ACC TGG CTG CTG TTG GTA CTG G-3’, was sub-cloned into EcoRI/KpnI sites of pCDNA3.1/Hisc A vector.

• pCDNA3.1/Hisc A Hck G2A.To produce this plasmid PCR frag- ment generated using pApuro Hck G2A as a template and the following primers: 5’-GCG AAT TCA TGG CCT GCA TGA AGT CCA AGT TCC-3’ and 5’-CGG GGT ACC TGG CTG CTG TTG GTA CTG G-3’, was sub-cloned into EcoRI/KpnI sites of pCDNA3.1/Hisc A vector.

• pCDNA3.1/Hisc A c-Src. To produce this plasmid PCR fragment generated using pApuro c-Src as a template and the following primers: 5’-G GAA TTC ATG GGT AGC AAC AAG AGC-3’ and 5’-CCC AAG CTT GAG GTT CTC CCC GGG CTG GTA C-3’, was sub- cloned into EcoRI/HindIII sites of pCDNA3.1/Hisc A vector.

• pCDNA3.1/Hisc A Lyn. To produce this plasmid PCR fragment generated using pApuro Lyn as a template and the following primers: 5’-G GAA TTC ATG GGA TGT ATA AAA TCA AA -3’ and 5’-CGG GGT ACC AGG CTG CTG CTG GTA TTG CCC TTC C-3’, was sub-cloned into EcoRI/KpnI sites of pCDNA3.1/Hisc A vector. • pCDNA3.1/Hisc A Fyn. To produce this plasmid PCR fragment

generated using pApuro Fyn as a template and the following primers: 5’-G GAA TTC ATG GGC TGT GTG CAA TGT-3’ and 5’-CGC GGA TCC CAG GTT TTC ACC AGG TTG GTA CTG-3’, was sub- cloned into EcoRI/BamHI sites of pCDNA3.1/Hisc A vector.

• pMSCV IRES EGFP Hck wt. To produce this plasmid Hck wt fragment was sub-cloned from pApuro Hck wt into EcoRI cloning site of pMSCV IRES EGFP vector.

• pMSCV IRES EGFP Hck K269R. To produce this plasmid Hck K269R fragment was sub-cloned from pCDpuro Hck K269R into EcoRI cloning site of pMSCV IRES EGFP vector

• pMSCV IRES EGFP Hck Y501F. To produce this plasmid Hck Y501F fragment was sub-cloned from pApuro Hck Y501F into EcoRI cloning site of pMSCV IRES EGFP vector.

• pCDHF3, gift of Dr. Hitoshi Kiyoi, Nagoya University School of Medicine, Nagoya, Japan

• pCDNA6A hFlt3 wt, gift of Dr. Spiekermann, University of Mu- nich, Germany (generated by BamHI/PstI sub-cloning of Flt3 wt frag- ment from pCDHF3 plasmid)

• pCDHFTD, contains a full-length human Flt3 ITD (Mt3) cDNA cloned from pCDSRα expression vector, gift of Dr. Hitoshi Kiyoi, Nagoya University School of Medicine, Nagoya, Japan [111]

Flt3 ITD fragment from pCDHFTD plasmid, gift of Dr. Spiekermann, University of Munich,Germany

• pCDNA6A c-kit, gift of Dr. Spiekermann, University of Munich, Germany

• pCDNA6A hFlt3 K644R,generated by mutagenesis • pCDNA6A hFlt3 Y589F,generated by mutagenesis • pCDNA6A hFlt3 Y591F,generated by mutagenesis

• pCDNA6A hFlt3 Y589F, Y591F,generated by mutagenesis • pCDNA6A hFlt3 Y589F, Y591F, Y597F, Y599F,generated by

mutagenesis

• pCDNA6A hFlt3 K644R, Y589F, Y591F,generated by mutage- nesis

• pCDNA6A hFlt3 K 644R, Y589F, Y591F, Y597F, Y599F, generated by mutagenesis

• pCDNA6A hFlt3 ITD K644R,generated by mutagenesis • pCDNA6A hFlt3 ITD Y589F,generated by mutagenesis • pCDNA6A hFlt3 ITD Y591F,generated by mutagenesis

• pCDNA6A hFlt3 ITD Y589F, Y591F,generated by mutagenesis • MIY hFlt3 wt. To produce this plasmid BamHI/PstI fragment en-

coding hFlt3 wt was sub-cloned into HpaI site of MIY vector.

• MIY hFlt3 ITD. To produce this plasmid BamHI/PstI fragment encoding hFlt3 ITD was sub-cloned into HpaI site of MIY vector.