Gene knockout by inducing P-element transposition in Drosophila

Abstract

ABSTRACT. The P-element has been successfully used in germline transformation to create transgenic flies and as an insertion mutagenesis agent to disrupt genes. Moreover, P-element can also be used to knockout genes that are near the insertion sites by inducing its imprecise transposition. In this article, P-element insertion lines were crossed with transposase expression fly to recover the transposon, and the deletion of flanking sequence, which was created by imprecise excision, was detected by PCR. Through this method, null alleles of seven genes that spread over three major chromosomes (X, second, third) were generated in Drosophila melanogaster. Results show that the frequency of flanking deletions is expected to be 1%, and it is enough to pick up at least one ideal deletion line from 200 independent recovery lines in general. These data suggest that although the frequency of disrupted gene varied greatly, from 0.13 to 2.34%, gene knockout by inducing P-element transposition appears to be a feasible and effective strategy, compared to the complicated process of gene targeting based on homologous recombination.

The First Defined Null Allele of the Notch Regulator, a Suppressor of Deltex: Uncovering Its Novel Roles in Drosophila melanogaster Oogenesis

“…To examine the full requirement for Su(dx), it is necessary to generate a defined null allele in which the complete open reading frame is deleted. In this study, we generated the first defined null allele, Su(dx) JD , using imprecise p-element excision [19]. We show that, in the wing and leg tissues, the null phenotype is essentially similar to the phenotypes reported for existing Su(dx) alleles, i.e., it demonstrates similar temperature-dependent outcomes and genetic interactions with dx and Notch alleles.…”

MicroRNA miR‐252 targets mbt to control the developmental growth of Drosophila

“…2A), following standard procedures (Ou, 2013). We first isolated a deletion allele referred to as miR-252 IM11 using a P-element strain, P{GT1} BG01009 , by inducing the imprecise excision of the P-element that is located 1845 bp upstream of the locus encoding the miR-252 precursor ( Fig.…”

“…Imprecise and precise excisions of the P-element were achieved by crossing the P{GT1} BG01009 strain with flies expressing Δ2-3 transposase (Ou, 2013) to generate lines w 1118 ; miR-252 IM11 and w 1118 ; Pre-Ex respectively. Imprecise and precise excisions of the P-element were achieved by crossing the P{GT1} BG01009 strain with flies expressing Δ2-3 transposase (Ou, 2013) to generate lines w 1118 ; miR-252 IM11 and w 1118 ; Pre-Ex respectively.…”

“…We first isolated a deletion allele referred to as miR-252 IM11 using a P-element strain, P{GT1} BG01009 , by inducing the imprecise excision of the P-element that is located 1845 bp upstream of the locus encoding the miR-252 precursor ( Fig. 2A), following standard procedures (Ou, 2013). The mutant allele contained the deletion of an approximately 2100 bp genomic region, including the miR-252 precursor-coding sequence ( Fig.…”

“…An miR-252 deletion mutation was generated using a P-element strain w 1118 ; P{GT1} BG01009 , which was obtained from the Bloomington Drosophila Stock Center. Imprecise and precise excisions of the P-element were achieved by crossing the P{GT1} BG01009 strain with flies expressing Δ2-3 transposase (Ou, 2013) to generate lines w 1118 ; miR-252 IM11 and w 1118 ; Pre-Ex respectively. The deletion breakpoints were determined by sequencing genomic DNA amplified by PCR using a pair of primers (Supporting Information Table S1).…”