Dual luciferase system to identify the most optimum mutant selective constructs.

Abstract

<p>Coding sequences for the first 17 amino acids of <i>KRAS</i> wild-type (wt) and mutant (mu) were inserted into the amino terminus of the psiCHECK2’s hRluc (renilla) and hluc (firefly) coding sequence, respectively. Knockdown of wt vs. mu sequence is compared by renilla to firefly intensity ratio. <b>A.</b> Schematic of the sequence insertion into psiCHECK2 for reporter constructs. <b>B.</b> Bar graph show comparison of reporter constructs relative light unit (RLU) intensity ratio of renilla (RL) to firefly (FF). Y-axis is RL/FF RLU ratio. X-axis is reporter test vectors and parent reporter vector. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. <b>C.</b> Positional effect of G12D knockdown constructs; panel <b>a</b>: Table illustrate each constructs guide strand sequence in relation to G12D mutation site. 1^st column indicates position of G12D mutation in guide strand of each construct. 2^nd column is the code for each construct. The guide strand sequence is shown as the complement of target sequence at 3’ to 5’ orientation; panel <b>b</b>: Bar graph show comparative plot of FF/RL RLU ratio (mu/wt) for each knockdown construct. Sample C is the control without knockdown vector. The red bar represents average control sample value for visual enhancement. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. Two-tailed student T-test indicates ρ-value ≤ 0.05 between control and samples 86, 87, 88, 75, 76 and 77. <b>D.</b> G12D, G12V, G12R and G12C knockdown constructs (with mutation nucleotide at position 3 or 4 of the guide strand) were tested against test reporter vectors of all four mutations. Bar graph shows the summary of relative average FF/RL RLU ratio (mu/wt). * indicate the most effective constructs for each mutation. X-axis is the knockdown constructs for G12D, G12V, G12R and G12C. P3 indicate knockdown construct with mutated nucleotide at position 3 of the guide strand. P4 indicate knockdown construct with mutated nucleotide at position 4 of the guide strand. Y-axis is the FF/RL RLU ratio.</p