To test whether a similar miRNA-associated regulation as let-7 miRNA regulates Lin28, we made a deletion series of the PpCSP1 3′-UTR fusing each fragment after the stop codon of the sGFP reporter gene driven by the constitutive rice Actin 1 promoter46,47 (Fig. 3h).
To study its functional role, we made a deletion mutant lacking the PxxP motifs between amino acid positions 468-486 (named ΔCTPxxP).
Sequencing of 20 clones demonstrated that the OAGR concatemer junction corresponded to the sequence of the oligo, effectively making a deletion that includes the ITR junction.
2) Similarly, given that ULK3 performs a checkpoint function via IST1 and CHMP4C (i.e. ULK3 is likely non-essential for growth and cell division), they should be also able to make a full deletion of ULK3 in cell lines to determine the precise phenotype.
To further test the effect of exon length, they make a library of deletions within exon 2 and measure circularization relative to deletion size, finding a very rough increase in circle formation with longer exons.
We were able to make such a deletion, however, using a two-cut strategy (strategy 4, experiment 25).
The specificity of the Cre-lox interaction also provides the basis for making a gene deletion in only certain cells.
To make a PTEN construct with deletion of the last six residues (TQITKV), PCR was conducted to introduce premature termination, using pcDNA3.1-PTEN as a template.
Our results, as well as those of others, show that insertions and deletions make a large contribution to the mutational landscape: The numbers of substituted, inserted, and deleted nucleotides per generation are all of the same order of magnitude.
Briefly, chromosomal abbreviations were calculated using the ADM2 statistic algorithm with a moving average window of 1 Mb; additionally, the default thresholds of ADM2 recommended by Agilent were used to make an amplification or deletion call.
