Data Link 4
Presented below is a more detailed version of plasmid constructions for recombinant DNA molecules used in:
Gilmore TD, C Cormier, J Jean-Jacques & M-E Gapuzan. (2001) Malignant transformation of primary chicken spleen cells by human transcription factor c-Rel. Oncogene 20: 7098-7103
Further details can be obtained by sending a request for information to Tom Gilmore at firstname.lastname@example.org.
Plasmid descriptions and constructions
· Plasmid pMH105 (v-Rel/Neo) was a kind gift of Mark Hannink (University of Missouri) and has been described previously (White & Gilmore, 1996).
· Plasmid c-Rel/Neo (human c-rel and neo) was constructed by subcloning an XbaI to SpeI fragment containing a full-length human c-rel cDNA into pMH105 which had been cleaved with XbaI.This excises v-rel and thus human c-rel replaces v-rel as the 5’ gene.
· The plasmid for only the expression of Bcl-2 was created by digesting plasmid v-R273H-bcl-2 (White & Gilmore, 1996) with XbaI and religating (this removes the 5’ mutant v-rel gene).
· Plasmid c-Rel/Bcl-2 was made by inserting the XbaI-SpeI fragment containing human c-rel (see above) at the XbaI site of the Bcl-2 (alone) expression plasmid.
· In plasmid c-Rel/Bcl-XL the human bcl-xL cDNA was used to replace the 3’ neo gene (removed by NcoI/RsrII digestion from pMH105 [i.e., v-Rel/Neo]).This created a v-Rel/Bcl-xL vector.This vector was then digested with XbaI (to remove the 5’ v-rel gene) and v-rel was replaced by an XbaI-SpeI fragment containing human c-rel.
· Plasmid c-Rel/mBcl-2 contains a mutant chicken bcl-2 gene, which is lacking the BH4 domain, and was made by digesting the bcl-2 gene with NaeI and SmaI and religating.These blunt-end enzymes result in the in-frame deletion of codons 4 to 33 of chicken bcl-2.
DW White & TD Gilmore (1996) Oncogene 13, 891-899.