Figure 2
Figure 2. Selective disruption of the translocated allele of MMSET by gene targeting in KMS-11 cells. (A) Schematic of the full-length MMSET exon structure. The targeting construct is designed to delete exon 7, which is shown in black. SA indicates splice acceptor; ARM, homology arm; IRES, internal ribosome entry site; NEO, neomycin resistance gene; and PA, polyadenylation signal. Triangles represent loxP sites. After Cre-loxP recombination, a single loxP site is left in place of the deleted exon 7. Splicing from exon 6 to exon 8 results in a reading frame shift and premature termination of translation after exon 6. (B) Detection of KMS-11 cells with targeted disruption of the translocated allele of MMSET. The translocation(4;14) generates fusion transcripts initiating in the IgH JH region on chromosome 14 that splice to exon 3 of MMSET. RT-PCR with primers A and B demonstrates fusion transcripts in parental KMS-11 cells, 2 clones with targeted deletion of MMSET exon 7, and a control clone with random integration of the targeting vector (bottom, lanes 1-4). RT-PCR with primers A and C indicate fusion transcripts spliced to the neomycin cassette, which can be generated only by targeting of the translocated MMSET allele (bottom, lane 8). MW indicates DNA size ladder. (C) Differential transcription of the translocated and nontranslocated MMSET alleles revealed by gene targeting. Q-PCR analysis for the intact MMSET I isoform was performed as described using cDNA from 2 random integrant control KMS-11 clones, a clone with disruption of the translocated MMSET allele (T-KO) and 2 clones with disruption of the nontranslocated allele (non-T-KO). Intact MMSET I was detected using an exon 7-specific forward primer. Normalized MMSET I transcript levels are expressed as a percentage of the average of the levels in the 2 random integrant clones. (D) Immunoblotting demonstrates extensive depletion of full-length MMSET protein in T-KO cells but not in non-T-KO cells. REIIBP protein levels are unaffected by deletion of MMSET exon 7, as would be predicted.

Selective disruption of the translocated allele of MMSET by gene targeting in KMS-11 cells. (A) Schematic of the full-length MMSET exon structure. The targeting construct is designed to delete exon 7, which is shown in black. SA indicates splice acceptor; ARM, homology arm; IRES, internal ribosome entry site; NEO, neomycin resistance gene; and PA, polyadenylation signal. Triangles represent loxP sites. After Cre-loxP recombination, a single loxP site is left in place of the deleted exon 7. Splicing from exon 6 to exon 8 results in a reading frame shift and premature termination of translation after exon 6. (B) Detection of KMS-11 cells with targeted disruption of the translocated allele of MMSET. The translocation(4;14) generates fusion transcripts initiating in the IgH JH region on chromosome 14 that splice to exon 3 of MMSET. RT-PCR with primers A and B demonstrates fusion transcripts in parental KMS-11 cells, 2 clones with targeted deletion of MMSET exon 7, and a control clone with random integration of the targeting vector (bottom, lanes 1-4). RT-PCR with primers A and C indicate fusion transcripts spliced to the neomycin cassette, which can be generated only by targeting of the translocated MMSET allele (bottom, lane 8). MW indicates DNA size ladder. (C) Differential transcription of the translocated and nontranslocated MMSET alleles revealed by gene targeting. Q-PCR analysis for the intact MMSET I isoform was performed as described using cDNA from 2 random integrant control KMS-11 clones, a clone with disruption of the translocated MMSET allele (T-KO) and 2 clones with disruption of the nontranslocated allele (non-T-KO). Intact MMSET I was detected using an exon 7-specific forward primer. Normalized MMSET I transcript levels are expressed as a percentage of the average of the levels in the 2 random integrant clones. (D) Immunoblotting demonstrates extensive depletion of full-length MMSET protein in T-KO cells but not in non-T-KO cells. REIIBP protein levels are unaffected by deletion of MMSET exon 7, as would be predicted.

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