a, Targeting of the intergenic miRNA cluster mir-290~295 replaces 2256bp of genomic sequence with the PGK-PuroΔtk cassette. The PGK-PuroΔtk deletion was by Cre recombinase transfection and FIAU selection. Alternatively RMCE by FLPo recombinase transfection was used to integrate a neomycin resistance (Neo^R ) plasmid (e.g. pMA_F3RoxNeoRoxTd-tomatoLoxPFRT). The PGK-Neo^R deletion was by Dre recombinase transfection. The predicted sizes of PCR products generated by PCR across the mir-290~295 cluster or modified alleles are indicated in brackets. b, Products for long-range PCR between primers specific for the selection cassette and the region external to the 5′ and 3′ homologous arms of the targeting vector. c, Products for PCR between primers specific for flanking sequences of mir-290~295 locus and the allelic variants. d, Bright field and corresponding fluorescent images of the same mir-290~295^Td-tomato/+ ES cell colony (top left), mir-290~295^PuΔtk/+ control ES cell colony (top right), embryoid bodies at d5 and d8 into the differentiation regime for mir-290~295^Td-tomato/+ (middle and bottom left) and mir-290~295^PuΔtk/+ (middle and bottom right). Scale bars are 100μm.

a. The mir-106a~363 cluster was removed as a 932bp deletion using the PuroΔtk cassette. The predicted sizes of PCR products generated by PCR (internal to the targeting vector) across the mir-106a-363 cluster or modified alleles are indicated in brackets. An RMCE cassette containing a cloned genomic sequence for the wildtype mir-106a~363 cluster (pMA_F3RoxNeoRox_miR106a~363_LoxPFRT) was inserted into the targeted locus by co-transfected with PGK-FlpO followed by FIAU and G418 selection. The PGK-Neo^R deletion was by Dre recombinase transfection. Cre deletion successfully deleted the conditional allele, although the mir-106a~363⁻ clone analysed in this figure was derived by Cre deletion of the mir-106a~363^PuΔtk clone. b, Long-range PCR between primers specific for the selection cassette and the region external to the 5′ and 3′ homologous arms of the targeting vector. c, PCR between primers (internal to the targeting vector) specific for flanking sequences of mir-106a~363 locus and the allelic variants. d, Comparative quantitative PCR for miRNAs within the mir-106a~363 cluster and miR-294 as a control. The TaqMan MicroRNA assays used are color coded. n=3; error bars show SD.

The Kanamycin resistance recombineering cassette was PCR amplified from the pHPmK3 vector and recombineered into BACs in E.coli deleting the miRNA gene. Retrieval of the homology arms and kanamycin selection cassette was achieved by recombineering a PCR amplified pPL611 vector backbone to create a PGK-Neo targeting vector (Neo-TV). The kanamycin/neomycin selection cassette of the targeting vector was switched to PuroΔtk by using a PGK-EM7-PuroΔtk-bpA restriction fragment for recombineering to generate the puromycin version of the targeting vector (Puro-TV).

a, The mir-21 gene was removed as a 191bp deletion using the PuroΔtk cassette which could then be a substrate for either deletion or RMCE using a reporter construct. The predicted sizes of PCR products generated by PCR across the mir-21 locus or modified alleles are indicated in brackets. b, Products for long-range PCR between primers specific for the selection cassette and the area external to the 5′ and 3′ homologous arms of the targeting vector. c, Products for PCR across the targeted and modified mir-21 locus. Note that the mir-21⁻ allele yields a PCR product which migrates as a doublet with the wild type allele/JM8.A3 PCR product. d, Bright field and fluorescent images of the same mir-21^Td-tomato/+ undifferentiated ES cell colonies (top left) mir-21^PuΔtk/+ control ES cell colonies (top right), embryoid bodies at d5 and d16 and d19 of differentiation for mir-21^Td-tomato/+ and mir-21^PuΔtk/+ as indicated. The bottom image shows a close-up of a d16 differentiation mir-21^Td-tomato/+ embryoid body. Scale bars are 100 μm.