Figure 5

Targetron donor plasmid and use of targetrons for conditional and non-conditional gene disruptions. (A) Targetron donor plasmid. The plasmid expresses a modified group II intron with the reverse transcriptase (RT) ORF deleted (I-ΔORF) and flanked by short exons under control of an active promoter (P_A), which can be either inducible or constitutive. The RT ORF is expressed in tandem from a location just downstream of E2. Protein-assisted splicing of the primary transcript produces a ribonucleoprotein (RNP) complex, which contains the group II intron RT bound to the excised intron lariat RNA and which promotes site-specific integration of the intron into DNA target sites via retrohoming (see Figure 3). (B) Use of targetrons for conditional and non-conditional gene disruptions. Conditional disruptions are obtained when the intron is targeted to insert into the top or sense strand of the target gene. Transcription of the target gene from its own promoter in the host chromosome (P_C) results in a primary transcript from which the intron can be removed by providing the RT, which promotes protein-assisted RNA splicing. Non-conditional disruptions are obtained by targeting of the intron to the bottom or antisense strand, which results in the insertion of the intron in an antisense orientation relative to that of the target gene. Transcription of the target gene then yields a primary transcript containing the complement of the intron, which is inactive and cannot be removed by RNA splicing.