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This pDV[cycC::cycC-CTAP] plasmid was transformed into Dictyostelium Ax2 cells and transformants selected by neomycin resistance to generate Ax2[cycC::cycC-CTAP] cells.
The constructs were introduced into Dictyostelium Ax2 cells by electroporation and transformants selected by growth in the presence of G418 (10 µg/ml) as the expression plasmids contain the neomycin resistance gene (neoR).
Some of these positive transformants selected by PCR were grown in liquid TAP medium and their carotenoids content analysed by HPLC and the relative mRNA levels of C. reinhardtii PSY (CrPSY) and CzPSY genes determined by qRT-PCR.
Stable ARL6 RNAi cell lines were also transfected with NotI digested pTbMYCBBS1, transformants selected by the addition of 10 μg/ml hygromycin and cell lines analysed as in Section 2.7.
For each transformation, 50 positive transformants were selected by tributyrin flat screening.
The transformants were selected by 5-FOA counter selection.
After incubation at 50 °C for 2 h, 5 μL was then used to transform BL21 Gold (DE3), and transformants were selected by being plated on LB agar containing 50 μg/mL kanamycin.
The transformants were selected by 5-Fluoroorotic Acid (5-FOA) counter selection.
Transformants were selected by plating seeds on MS plates containing 20 mg/l hygromycin B. The positive transformants were confirmed by PCR using primers HPT-F (5'-cttctgcgggcgatttgtgt-3') and HPT-R (5'-ttcgatgtaggagggcgtgga-3') designed specifically for the hygromycin resistance gene in this vector.
Plants were transformed by the floral dip method [ 31], and transformants were selected by spraying with 6 X 10-3% glufosinate ammonium herbicide.
Transformants were selected by planting the seeds on 1/2 MS plates containing 50 mg/L hygromycin B. The positive transformants were confirmed using PCR.
