Figure 1.
Figure 1. Knock-in of the conditional MN1-TEL transgene into the mouse Aml1 locus. (A) Scheme representing the mouse Aml1 genomic locus, the targeting vector, the conditional MN1-TEL knock-in (KI) allele generated by homologous recombination, and the activated allele after Cre-mediated deletion of the transcriptional stop cassette. The Aml1 exons 3 and 4 are indicated by solid boxes. The human AML1 cDNA (62 bp of exon 4 followed by the remaining coding region in exons 5 to 7) was inserted in-frame into mouse Aml1 exon 4 to maintain AML1 expression from the KI allele. We flanked a cassette containing a transcriptional strong stop followed by a phosphoglycerokinase promoter (open box)–driven neomycin resistance gene (Neor) with lox-P recombination sites. An IRES (internal ribosome entry site)–MN1-TEL-IRES-GFP (green fluorescent protein)–polyA (polyadenylation sequence) cassette was cloned 3′ of the stop/Neor sequences. For negative selection, a diphtheria toxin-A cassette (DTA) was added. Hybridization probes to detect the targeted Aml1 allele (5′ probe) and removal of the stop/Neo cassette (IRES probe) are shown. (B) Southern blot detection of the Aml1-stop-IRES-MN1-TEL-IRES-GFP allele. Genomic DNA of ES cells (ES) or mice (M) was digested with XbaI and probed with the Aml1 5′ probe. WT indicates wild-type; KI, knock-in. (C) Southern blot detection of stop/Neo cassette removal. Southern blot showing XbaI-digested genomic DNA of tissues of MN1-TELKI/WT/Mx1-Cre+/WT (MN1-TEL/Mx1-Cre) mice injected with polyinosinic-polycytidylic acid (pI-pC) or not after hybridization with an IRES probe. This Southern blot yielded 8- and 8.5-kb bands (not separated in this figure) without Cre recombination while 8.5- and 5.5-kb bands were identified after recombination of the KI allele. Numbers below the lanes indicate the estimated efficiency of recombination. S indicates spleen; L, liver; BM, bone marrow; T, thymus. (D) GFP expression in MN1-TEL/Mx1-Cre mice. GFP expression was analyzed by FCM analysis 2 weeks after the last injection of pI-pC. Numbers indicated are mean ± SE percentage of GFP-positive cells (n = 5). (E) Detection of MN1-TEL protein in MN1-TEL/Mx1-Cre mouse BM cells. Western blot of BM cells probed with a TEL C-terminal antibody 2 weeks after the last injection with pI-pC or control phosphate-buffered saline (PBS). Gapdh detection was used as a loading control.

Knock-in of the conditional MN1-TEL transgene into the mouse Aml1 locus. (A) Scheme representing the mouse Aml1 genomic locus, the targeting vector, the conditional MN1-TEL knock-in (KI) allele generated by homologous recombination, and the activated allele after Cre-mediated deletion of the transcriptional stop cassette. The Aml1 exons 3 and 4 are indicated by solid boxes. The human AML1 cDNA (62 bp of exon 4 followed by the remaining coding region in exons 5 to 7) was inserted in-frame into mouse Aml1 exon 4 to maintain AML1 expression from the KI allele. We flanked a cassette containing a transcriptional strong stop followed by a phosphoglycerokinase promoter (open box)–driven neomycin resistance gene (Neor) with lox-P recombination sites. An IRES (internal ribosome entry site)–MN1-TEL-IRES-GFP (green fluorescent protein)–polyA (polyadenylation sequence) cassette was cloned 3′ of the stop/Neor sequences. For negative selection, a diphtheria toxin-A cassette (DTA) was added. Hybridization probes to detect the targeted Aml1 allele (5′ probe) and removal of the stop/Neo cassette (IRES probe) are shown. (B) Southern blot detection of the Aml1-stop-IRES-MN1-TEL-IRES-GFP allele. Genomic DNA of ES cells (ES) or mice (M) was digested with XbaI and probed with the Aml1 5′ probe. WT indicates wild-type; KI, knock-in. (C) Southern blot detection of stop/Neo cassette removal. Southern blot showing XbaI-digested genomic DNA of tissues of MN1-TELKI/WT/Mx1-Cre+/WT(MN1-TEL/Mx1-Cre) mice injected with polyinosinic-polycytidylic acid (pI-pC) or not after hybridization with an IRES probe. This Southern blot yielded 8- and 8.5-kb bands (not separated in this figure) without Cre recombination while 8.5- and 5.5-kb bands were identified after recombination of the KI allele. Numbers below the lanes indicate the estimated efficiency of recombination. S indicates spleen; L, liver; BM, bone marrow; T, thymus. (D) GFP expression in MN1-TEL/Mx1-Cre mice. GFP expression was analyzed by FCM analysis 2 weeks after the last injection of pI-pC. Numbers indicated are mean ± SE percentage of GFP-positive cells (n = 5). (E) Detection of MN1-TEL protein in MN1-TEL/Mx1-Cre mouse BM cells. Western blot of BM cells probed with a TEL C-terminal antibody 2 weeks after the last injection with pI-pC or control phosphate-buffered saline (PBS). Gapdh detection was used as a loading control.

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