Engineering Inducible Knock-In Mice to Model Oncogenic Brain Tumor Mutations from Endogenous Loci

Abstract

To maximize the physiological relevance of in vivo brain tumor mouse models designed to study the downstream effects of oncogenic mutations, it is important to express the mutated genes at appropriate levels, in relevant cell types, and in the proper developmental context. For recurrent mutations found in the heterozygous state in tumors, expression of the mutation from the endogenous locus is a more physiologically relevant recapitulation of the brain tumor genome. Here, we describe an approach to generate knock-in mice with an inducible mutation recombined into the endogenous locus. In these engineered mice, the mutated allele is designed for expression controlled by the endogenous promoter and regulatory elements after Cre recombinase-mediated deletion of a loxP-STOP-loxP cassette inserted upstream of the translational start site. To preserve the structure of the endogenous locus, mutations or additional elements may need to be inserted at a considerable distance from the loxP-STOP-loxP cassette. We used recombineering to build a construct with two selectable markers and multiple genetic alterations that can be introduced into the endogenous allele in cis with a single ES cell targeting.

Keywords: Brain tumor; Cre recombinase; Knock-in; Mouse; Mutation; Neuro-oncology; Oncogene; Recombineering; Transgenic; loxP.

Fig. 1.. Designing An Inducible H3f3a Knock-In Vector.

(a) Schematic map of the H3f3a gene locus defining intron and exon configuration, endogenous translation initiation (green arrow, exon 2) and stop (red hexagon, exon 4) sites. (b) Final design of targeted knock-in H3f3a locus illustrating important engineering features including intron 1 placement ofloxP-STOP-loxP (STOP) transcription termination sequence that will also provide puromycin (Puro) positive selection in ES cells, exon 2 point mutation (red asterisk), exon 4 in-frame epitope tag (yellow tag) associated with intron 3 placement of Frt-Neo-Frt cassette to provide G418 positive selection in ES cells. loxP (cyan) Frt (yellow) and FrtF3 (yellow striped) sequences are marked with triangular arrowheads. Dotted line shows genomic locus not included in targeting construct. EcoRI/BamHI sites and SacI sites generate restriction fragments detected by 5′ and 3′ probes, respectively, to create distinguishable Southern blot DNA fragment sizes before and after targeting, with a SacI site designed into the Frt-Neo-Frt cassette cloning. Probes are designed to hybridize outside homologous recombination events for proper Southern blot verification. (c) Final design of H3f3a NotI-linearized targeting vector. Dotted blue line indicates targeting vector backbone. Vector backbone provides Diphtheria toxin (DT) negative selection in ES cells. Short (2 kb) and long (4.6 kb) homology arms flank engineered locus (17.5 kb) to guide homologous recombination outside of engineered elements. Not drawn to scale so that elements can be clearly marked.

Fig. 2.. H3f3a Locus Retrieval.

(a) Schematic of desired 16.2 kb H3f3a locus including exons 1–4 within a 124 kb BAC clone. Dotted black line indicates genomic regions outside of region to be retrieved. (b) Retrieval vector with specific locus homology retrieval arms (5′ Ret and 3′ Ret), linearized with HindIII and SpeI to facilitate gap repair homologous recombination. pBR322-DT Vector (blue) is equipped to express Diphtheria toxin (DT) for negative selection in mammalian cells and ampicillin resistance (Amp) in bacterial cells. (c) Successfully retrieved locus. EagI sites mark diagnostic digest points to verify vector fidelity. PacI and NsiI sites flank exon 2 facilitating mutant exon 2 replacement. (d) Retrieved locus with successfully replaced mutated exon 2 (red asterisk). If mutation in the gene of interest is located distally to the loxP-STOP-loxP cassette, it would be introduced by recombineering as a distal element in Targeting 2 rather than at this early stage.

Fig. 3.. Assembling Targeting 1 Vector For loxP-STOP-loxP Insertion.

(a) Partial targeting 1a vector containing subcloned XhoI-flanked H3f3a intron 1 5′ homology targeting arm (5′ Tar) and loxP-STOP-loxP (STOP) cassette equipped to express puromycin (Puro) in mammalian cells. EagI is the designed diagnostic subcloning remnant restriction enzyme site. (b) Partial targeting 1b vector containing subcloned XhoI/SalI and NotI-flanked Frt-Neo-Frt (Neo) cassette equipped to express neomycin in bacterial and mammalian cells, and H3f3a intron 1 3′ targeting homology arm (3′ Tar). BamHI is a subcloning remnant restriction enzyme site. (c) Final targeting 1 vector. loxP sites (cyan arrowheads), Frt sites (yellow arrowheads), ampicillin resistance in bacterial cells (Amp).

Fig. 4.. Targeting 1: Inserting loxP-STOP-loxP.

(a) Isolated targeting 1 cassette excised with XhoI and NotI to facilitate gap repair homologous recombination. (b) Intermediate step of targeting 1: Successful insertion of targeting 1 loxP-STOP-loxP/Neo selection cassette (5.9 kb) into H3f3a intron 1. EagI sites mark diagnostic digest points to verify vector fidelity. (c) Successful Flippase excision of Neo selection cassette leaving behind a remnant Frt site and stable loxP-STOP-loxP. EagI sites mark diagnostic digest points to verify vector fidelity.

Fig. 5.. Assembling Targeting 2 Vector For Distal Element Insertion.

(a) Partial targeting 2a vector containing subcloned HindIII and EcoRI-flanked H3f3a intron 3 5′ targeting homology arm (5′ Tar). (b) Partial targeting 2b vector containing subcloned HindIII/EcoRI and SacII-flanked Frt-Neo-Frt (Neo) cassette to express neomycin in bacterial and mammalian cells and H3f3a intron 3/exon 4 3′ targeting homology arm (3′ Tar). SacI is the designed subcloning remnant restriction enzyme site for Southern blot verification. (c) Final targeting 2 vector. Mutated Frt sites (striped yellow). 3′ Tar is engineered to contain the diagnostic linker EagI restriction enzyme site and epitope tag sequence.

Fig. 6.. Targeting 2: Inserting Distal Element.

(a) Targeting 2 cassette fragment isolated with HindIII and SacII to facilitate gap repair homologous recombination. (b) Final H3f3a knock-in targeting vector generated by successful insertion of targeting 2 Neo selection/distal element cassette (2.3 kb) into H3f3a intron 3/exon 4. EagI sites mark diagnostic digest points to verify final gene targeting vector fidelity.

Fig. 7.. Verifying H3f3a Knock-In Targeting Vector Construction.

EagI restriction enzyme digests to verify correct assembly for each step of H3f3a knock-in targeting vector construction.