FLP-mediated conditional loss of an essential gene to facilitate complementation assays Page: 48
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CmGAS1, AtSUC2) was cloned into the Sbfl and EcoRI sites of plasmid pCAM-Gent-EASE
FLP, generating the series of plasmids pCAM-Gent-TSpSUC2-EASE-FLP. The plasmids
pGPTV-RolCpSUC2, pGPTV-CoYMVpSUC2 and pGPTV-GASpSUC2 (Srivastava, Ganesan,
Ismail, Ayre, manuscript in preparation) were digested with restriction enzymes Sbfl and EcoRI
generating the TSpSUC2 cassettes (promoter-SUC2 cDNA-polyadenylation signal) (1)
RolCpSUC2, (2) CoYMVpSUC2, and (3) GASpSUC2, respectively. Each of the above cassettes
was then cloned into the Sbfl/EcoRI digested pCAM-Gent-EASE-FLP as backbone, generating
the vectors (1) pCAM-Gent-RolCpSUC2-EASE-FLP, (2) pCAM-Gent-CoYMVpSUC2-EASE-
FLP, and (3) pCAM-Gent-GASpSUC2-EASE-FLP. The plasmids were transformed into E. coli,
plasmid DNA was isolated and positive clones were identified by restriction analysis.
The vector pCAM-Gent-SUC2pSUC2-EASE-FLP with the AtSUC2 gene under the
control of its native promoter (SUC2p) was generated by digesting the plasmid pGPTV-
SUC2pSUC2 with restriction enzymes Sbfl and KpnI to obtain the SUC2p fragment. The SUC2p
was then cloned into the Sbfl and KpnI digested pCAM-Gent-RolCpSUC2-EASE-FLP as
backbone, replacing the rolCp. The vector was transformed into E. coli, plasmid DNA was
isolated and positive clones were identified by restriction analysis.The plasmids pCAM-Gent-
RolCpSUC2-EASE-FLP, pCAM-Gent-CoYMVpSUC2-EASE-FLP, pCAM-Gent-GASpSUC2-
EASE-FLP and pCAM-Gent-SUC2pSUC2-EASE-FLP were then transformed into
Agrobacterium tumefaciens strain GV3101mp90 by electroporation.
3.11 Plant Transformation
Plant transformation was performed by the floral-dip method (Clough and Bent, 1998).
Arabidopsis thaliana plants were grown in 3.5 inch square pots, in a plant growth chamber under48
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Ganesan, Savita. FLP-mediated conditional loss of an essential gene to facilitate complementation assays, thesis, December 2007; Denton, Texas. (https://digital.library.unt.edu/ark:/67531/metadc5180/m1/58/: accessed July 16, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; .