A conditional knockout resource for the genome-wide study of mouse gene function

Abstract

Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.

Figure 1. Schematic of the ‘knockout-first’ conditional allele

The ‘knockout-first’ allele (tm1a) contains an IRES:lacZ trapping cassette and a floxed promoter-driven neo cassette inserted into the intron of a gene, disrupting gene function. Flp converts the ‘knockout-first’ allele to a conditional allele (tm1c), restoring gene activity. Cre deletes the promoter-driven selection cassette and floxed exon of the tm1a allele to generate a lacZ-tagged allele (tm1b) or deletes the floxed exon of the tm1c allele to generate a frameshift mutation (tm1d), triggering nonsense mediated decay of the deleted transcript.

Figure 2. Computational design of oligonucleotides for recombineering and LR-PCR genotyping

a, A critical exon(s) common to all transcript variants (red box) is identified. Recombineering oligonucleotides (50-mers) are identified by ArrayOligoSelector within pre-defined blocks (G5, U, D, G3) of genomic sequence for insertions of the targeting cassette and 3′ loxP site and for plasmid rescue of the 5′ and 3′ homology arms by gap repair. For LR-PCR genotyping, multiple primers (25 to 30-mers) are then selected from 1-kb blocks of genomic sequence (GF, GR) outside the homology arms. b, Display of conditional alleles on the Ensembl genome browser (Distributed Annotation System (DAS) source = KO alleles). A conditional design for the merged Ensembl/Havana Rbmx gene on the reverse strand is shown.

Figure 3. Construction of Gateway-adapted intermediate targeting vectors by 96-well BAC recombineering

Recombineering steps and elapsed time are shown. a, BAC clones, arrayed in 96-well format and electroporated with a plasmid expressing arabinose-inducible Red proteins (pBADgbaA). b–d, After arabinose induction, cells are electroporated with PCR fragments containing R1-pheS/zeo-R2 Gateway element (b), loxP-kan-loxP cassette (c) and R3-ori/ampR-R4 subcloning plasmid (d). e, After gap repair, plasmid DNA is prepared and transformed into Cre-expressing bacteria to remove the kanR cassette, leaving a single loxP site downstream of the critical exon. Antibiotics used at each step are: A, ampicillin; C, chloramphenicol; K, kanamycin; T, tetracycline; Z, zeocin.

Figure 4. Intermediate and final targeting constructs

a, Schematic showing the structure of the Gateway-adapted intermediate plasmid. A rare AsiSI restriction site is included in the gap repair plasmid for linearizing the final targeting vector before electroporation of ES cells. b, Assembly of final targeting vectors in a multi-Gateway reaction. See Supplementary Fig. 4 for a full description of the custom Gateway-adapted plasmids used for vector construction.

Figure 5. Genotyping ES clones by LR-PCR sequencing

Five LR-PCR reactions are carried out: two 5′ arm (GF/5′U), two 3′ arm (3′U/GR) and one cassette (3′U/LX). Sequence verification of LR-PCR products is carried out with gene-specific primers (GF and GR) and with nested primers in the targeting cassette (5′Us and 3′Us). To confirm the presence or absence of the 3′ loxP site, 3′ arm LR-PCR products are sequenced with a primer adjacent to the loxP site (LR). In cases where 3′ arm LR-PCR fails to generate a product, the 3′ loxP site is confirmed by sequencing the cassette product.