![Figure 1. BCR-ABL1 kinase and the TKI-resistant T315I mutant promote HomeoRR in a “dosage”-dependent manner. (A) Homeologous recombination cassette contains a functional hygromycin gene (hyg), used to select for stably transfected cells, and a 3.9-kb tk-neo fusion gene rendered nonfunctional by the insertion of a 22-bp oligonucleotide containing 18-bp recognition site for endonuclease I-SceI (yellow strip). Three AluI restriction sited (red stripes) are present, too. In addition, a 2.5-kb fragment containing a complete HSV-1 tk gene, which is more than 1% divergent compared with that in the tk-neo fusion gene (0.8% and 1.5% divergent in 5′ and 3′ direction from the DSB site, respectively), is inserted. This inserted fragment serves as a potential recombination template (tk donor). HomeoRR by gene conversion between the I-SceI–induced DSB in the tk-neo fusion gene and the divergent tk donor template restores the function of neo gene encoding resistance to G418 (G418-R) and removes 2 AluI restriction sites closest to the DSB site.25 (B) Southern blot analysis of 32Dcl3-pLB4 parental cell clone (P) and BCR-ABL1–32Dcl3-pLB4 clone (B/A), in which a single copy of the pLB4 cassette was integrated in the genome. Genomic DNA was digested with HindIII and blotted with radiolabeled NcoI-NarI fragment of neo gene as a probe. (C) The 32Dcl3-pLB4 parental cells (P), cells expressing nonmutated BCR-ABL1 (B/A[nm]) and BCR-ABL1(T315I) (B/A[T315I]), and also cells expressing low (B/A-low) and high (B/A-high) levels of BCR-ABL1 were cotransfected with I-SceI (DSB inducer) and GFP (transfection control) expression vectors. HomeoRR activity is shown as percentage of G418-resistant cells in the GFP+ population. (D) Representative analysis of the recombination products by PCR (the primer positions are indicated by blue arrows in panel A) followed by AluI restriction digest. Left box: Nonrecombinant cassette produces 1393-bp product, which generates 653-, 381-, 337-, and 22-bp bands when digested by AluI. Right box: HomeoRR was confirmed in the mixture of G418-resistant clones by PCR amplifying 1371-bp HomeoRR products, which after AluI restriction digest generate characteristic recombination-specific 990-bp band and also 381-bp band (panel A, scheme of the cassette).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/118/4/10.1182_blood-2010-09-307256/4/zh89991175380001.jpeg?Expires=1724269536&Signature=v4kcGlJWyyb9Qdt~eMbyo-KW9gn~sRdmE40QmBhHD1~wgdRj1lQO7Ot9mKkANnxXTVF1jm0PynKawlurBbk9Yaxbr5~iyhUtyFDD5sF9KqMhiXNb~U9Aib~HLkEisaF1bof3N-98bb0Ne0-Eg4pgRdRi9g1ByE1CJdauHz58qSrSj7oWWQLiv12FA9aksPft1YMPS2BUGgjHMFFC2L2KPS63Z652CwWlJWDGDU0zZYlni3VQaTB9ureqaCigi2F9CwvpG3OhxBXdRq8QPVSn0sn7r0HmioqVCgHMullMh81ADUNn~Z-l-eIUHQKGF6b3asFwITZzHxNekpXxmL2uDA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
BCR-ABL1 kinase and the TKI-resistant T315I mutant promote HomeoRR in a “dosage”-dependent manner. (A) Homeologous recombination cassette contains a functional hygromycin gene (hyg), used to select for stably transfected cells, and a 3.9-kb tk-neo fusion gene rendered nonfunctional by the insertion of a 22-bp oligonucleotide containing 18-bp recognition site for endonuclease I-SceI (yellow strip). Three AluI restriction sited (red stripes) are present, too. In addition, a 2.5-kb fragment containing a complete HSV-1 tk gene, which is more than 1% divergent compared with that in the tk-neo fusion gene (0.8% and 1.5% divergent in 5′ and 3′ direction from the DSB site, respectively), is inserted. This inserted fragment serves as a potential recombination template (tk donor). HomeoRR by gene conversion between the I-SceI–induced DSB in the tk-neo fusion gene and the divergent tk donor template restores the function of neo gene encoding resistance to G418 (G418-R) and removes 2 AluI restriction sites closest to the DSB site.25 (B) Southern blot analysis of 32Dcl3-pLB4 parental cell clone (P) and BCR-ABL1–32Dcl3-pLB4 clone (B/A), in which a single copy of the pLB4 cassette was integrated in the genome. Genomic DNA was digested with HindIII and blotted with radiolabeled NcoI-NarI fragment of neo gene as a probe. (C) The 32Dcl3-pLB4 parental cells (P), cells expressing nonmutated BCR-ABL1 (B/A[nm]) and BCR-ABL1(T315I) (B/A[T315I]), and also cells expressing low (B/A-low) and high (B/A-high) levels of BCR-ABL1 were cotransfected with I-SceI (DSB inducer) and GFP (transfection control) expression vectors. HomeoRR activity is shown as percentage of G418-resistant cells in the GFP+ population. (D) Representative analysis of the recombination products by PCR (the primer positions are indicated by blue arrows in panel A) followed by AluI restriction digest. Left box: Nonrecombinant cassette produces 1393-bp product, which generates 653-, 381-, 337-, and 22-bp bands when digested by AluI. Right box: HomeoRR was confirmed in the mixture of G418-resistant clones by PCR amplifying 1371-bp HomeoRR products, which after AluI restriction digest generate characteristic recombination-specific 990-bp band and also 381-bp band (panel A, scheme of the cassette).
