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Transgenic Research

, Volume 18, Issue 2, pp 261–279 | Cite as

A caveat in mouse genetic engineering: ectopic gene targeting in ES cells by bidirectional extension of the homology arms of a gene replacement vector carrying human PARP-1

  • Aswin Mangerich
  • Harry Scherthan
  • Jörg Diefenbach
  • Ulrich Kloz
  • Franciscus van der Hoeven
  • Sascha Beneke
  • Alexander BürkleEmail author
Original Paper

Abstract

Here we report an approach to generate a knock-in mouse model using an ‘ends-out’ gene replacement vector to substitute the murine Parp-1 (mParp-1) coding sequence (32 kb) with its human orthologous sequence (46 kb). Unexpectedly, examination of mutant ES cell clones and mice revealed that site-specific homologous recombination was mimicked in three independently generated ES cell clones by bidirectional extension of the vector homology arms using the endogenous mParp-1-flanking sequences as templates. This was followed by adjacent integration of the targeting vector, thus leaving the endogenous mParp-1 locus functional. A related phenomenon termed ‘ectopic gene targeting’ has so far only been described for ‘ends-in’ integration-type vectors in non-ES cell gene targeting. We provide reliable techniques to detect such ectopic gene targeting which represents an unexpected caveat in mouse genetic engineering that should be considered in the design and validation strategy of future gene knock-in approaches.

Keywords

ES cells Gene targeting Homologous recombination Knock-in mice PARP-1 

Abbreviations

DAPI

4′,6-Diamidino-2-phenylindol-dihydrochlorid

DSB

Double strand break

DTA

Diphtheria toxin A

ESC

Embryonic stem cell clone

F

Filial generation

FISH

Fluorescence in situ hybridization

hPARP-1

Human PARP-1

HR

Homologous recombination

mParp-1

Murine Parp-1

NeoR

Neomycin resistance

PARP-1

Poly(ADP-ribose) polymerase-1

qPCR

Quantitative PCR

SDSA

Synthesis-dependent strand annealing

wt

Wild-type

Notes

Acknowledgments

We thank Oliver Popp, Gudrun von Scheven, Daniela Gassen, Heidi Henseleit, and Birgitt Planitz for technical help and support, and Prof. Andrew Smith (Edinburgh, UK) for valuable discussion of the data. AM was supported by the ‘Studienstiftung des Deutschen Volkes’ and the ‘Deutsche Forschungs Gemeinschaft’ (DFG) (International Research Training Research Group 1331).

Supplementary material

11248_2008_9228_MOESM1_ESM.tif (1.1 mb)
Fig. S1 Schematic representation of the gene targeting strategy for the generation of human PARP-1 (hPARP-1) knock-in mice. An ‘ends-out’ gene-replacement targeting vector of 64 kb spanning the entire hPARP-1 coding sequence (46 kb) including all 23 exons and 22 introns and flanked by 5 kb of the murine regulatory sequences, i.e., murine Parp-1 (mParp-1) promoter and terminator, was constructed to replace the mParp-1 coding sequence with its orthologous human counterpart. A neomycin resistance (Neo R ) cassette was included in intron 14 and a diphtheria toxin A (DTA) cassette fused to the homologous terminator sequences to enable positive-negative selection of ES cell clones. (TIFF 1111 kb)
11248_2008_9228_MOESM2_ESM.tif (5 mb)
Fig. S2 Verification of Pfl23II linearization of the targeting vector by digestion with restriction enzymes XmaIII and NotI. (a) Schematic of the targeting vector showing recognition sites of relevant restriction enzymes. (b left) The Pfl23II-linearized targeting vector (64 kb) was visualized by 0.5% agarose gel electrophoresis and subsequent ethidium bromide staining. (b middle and right) The Pfl23II-linearized targeting vector was digested with XmaIII and NotI and fragments were separated by 0.8% agarose gel electrophoreses and visualized by ethidium bromide staining. Expected fragment sizes for the XmaIII digestion (b middle) in the case of complete Pfl23II digestion are the following: 35.94 kb, 17.18 kb, 5.27 kb, 3.42 kb, 2.06 kb, 25 bp. Please note the absence of a fragment of 8.68 kb, which should be visible in the case of incomplete Pfl23II digestion. Due to its small size, the 25 bp fragment is not visible in the gel. Expected fragment sizes for NotI digestion (b right) in the case of complete Pfl23II digestion are the following: 53.14 kb, 5.26 kb, 5.48 kb. Please note the absence of a fragment of 10.74 kb, which should be visible in the case of incomplete Pfl23II digestion. The two fragments of 5.26 kb and 5.48 kb could not be resolved and are visible as one band. The “cloudy” appearance of DNA below the Pfl23II-linearized targeting vector control lane is due to overloading of the gel with DNA (1.8 µg). L, molecular weight ladder (TIFF 5135 kb)
11248_2008_9228_MOESM3_ESM.tif (7.9 mb)
Fig. S3 Sequencing of flanking PCR amplicons (see Fig. 1) demonstrated specificity of the PCRs and showed bona fide site-specific homologous recombination in ES cell clones #113, #225, and #267. mParp-1 indicates the sequence of the endogenous murine Parp-1 locus; hPARP-1 ki, the sequence of the expected correct human PARP-1 knock-in locus (TIFF 8073 kb)
11248_2008_9228_MOESM4_ESM.tif (882 kb)
Fig. S4 Validation of hPARP-1 germline transmission. Flanking PCRs for promoter and terminator homology arms as shown in Fig. 1a using genomic DNA isolated from 16 hPARP-1 mice (F1), which were genotyped positive for the presence of hPARP-1. ESC, ES cell clone; F1, first filial generation; L, molecular size ladder (TIFF 881 kb)
11248_2008_9228_MOESM5_ESM.tif (3 mb)
Fig. S5 Validation of quantitative real-time PCR. (a) Quantitative real-time PCR using a dilution series with 10 ng to 200 ng of genomic DNA isolated from a heterozygous hPARP-1 mouse (PCRs for hPARP-1 and Cygb) or a wild-type mouse (PCRs for Lin9 and Gm821). Samples with 200 ng DNA were used as a standard and 2-ΔΔCt values of these samples were set to 1.0. (b) Validation of results from qPCR by test breeding. Potential homozygous hPARP-1 mice of lines #113 and #225 carrying two copies of hPARP-1 were bred to wild-type C57BL/6 (B6) mice. As expected by Mendelian law, resulting offspring (2xhPARP-1 x B6) was 100% mutant (2 representative litters out of 5) (TIFF 3036 kb)

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Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Aswin Mangerich
    • 1
  • Harry Scherthan
    • 2
  • Jörg Diefenbach
    • 1
  • Ulrich Kloz
    • 3
  • Franciscus van der Hoeven
    • 3
  • Sascha Beneke
    • 1
  • Alexander Bürkle
    • 1
    Email author
  1. 1.Molecular Toxicology Group, Department of BiologyUniversity of KonstanzConstanceGermany
  2. 2.Bundeswehr Institute of RadiobiologyMunichGermany
  3. 3.German Cancer Research Center, Transgenic Core FacilityHeidelbergGermany

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