Similarly, although some studies have used multiple mutant alleles, transgene rescue experiments to eliminate off-target effects are lacking, unless indicated otherwise.
The experiments are very rigorous and thorough, employing multiple mutant alleles, RNAi lines, transgenes, antibodies and biochemical reagents.
There was some tendency of the rescuants expressing wild-type endoA transgenes to withstand tetanic depression better than those expressing mutant transgenes (Figure 5A).
Similarly, ubiquitous expression of two mutant transgenes with the C-tail is lethal, while expression of three transgenes without the tail is not.
All lines displayed equivalent levels of rescue, in the case of wild-type transgenes, or lack of rescue in the case of mutant transgenes (Figure S1).
As demonstrated by Aslanidi et al., a multiple mutant of AAV2 led to ~24-fold higher efficiency in in vitro gene transduction, which was mainly because the combined mutants had better ability for escaping ubiquitination then resulting in more nuclear translocation and more transgene expression [ 30].
None of the mle mutant transgenes was able to rescue mle 1 homozygous males (Table 1).
To reduce the influence of this position effect, we generally tested multiple integration lines for the same mutant transgene (Figure 2).
However, severe DCM developed with the addition of a transgene encoding a DCM mutation, suggesting that the mutant transgene encodes a protein with dominant negative activity.
The application of this screen led to the identification of multiple mutants, which exhibit some distinct phenotypes relative to one another and to previously identified maize mutants that can reactivate the silent transgene.
