- Email: [email protected]
Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones
YABIO 11951
No. of Pages 3, Model 5G
12 February 2015 Analytical Biochemistry xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones
8
Ewelina Elert-Dobkowska a,b,1, J. Christopher Hennings c,1, Christian A. Hübner c, Christian Beetz a,⇑
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12 1 2 4 7 15 16 17 18 19 20 21 22 23 24 25 26
a
Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, 07747 Jena, Germany Department of Genetics, Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland c Institute of Human Genetics, Jena University Hospital, 07747 Jena, Germany b
a r t i c l e
i n f o
Article history: Received 27 October 2014 Received in revised form 9 January 2015 Accepted 12 January 2015 Available online xxxx Keywords: Embryonic stem cells Genome engineering Knockout MLPA Mouse model
a b s t r a c t Following locus-specific genome editing of mouse embryonic stem cells (ESCs), the identification of correctly targeted clones remains a challenge. We applied multiplex ligation-dependent probe amplification (MLPA) to screen for homologous recombination-based genomic integration of a knockout construct in which part of a gene is deleted. All candidate ESCs thereby identified were subsequently validated by conventional methods. Thus, MLPA represents a highly reliable as well as cost- and time-efficient alternative to currently applied methods such as Southern blotting and polymerase chain reaction (PCR)-based approaches. It is also applicable to knockin recombination strategies and compatible with the CRISPR/Cas9 system and other genome editing strategies. Ó 2015 Elsevier Inc. All rights reserved.
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Genetic engineering of model organisms has opened new opportunities for understanding gene function and thereby revolutionized many research areas. From a biomedical perspective, the editing of the mouse genome has proven to be most powerful. There are two fundamentally differing approaches for genotypedriven mutagenesis of murine embryonic stem cells (ESCs).2 One is the random insertion of a universal sequence cassette into the genome followed by complementary DNA (cDNA)-based identification of the desired intragenic events (‘‘gene trapping’’ [1]). The other is the alteration of predefined loci either by homologous recombination with exogenously provided DNA [2] or by the combination of site-specific nucleases with a DNA-modifying repair process [3]. In these locus-specific methods, a varying proportion of ESC clones obtained does not carry the intended alteration. Identification of correctly targeted clones (often accounting for <1%) requires analysis of their genomic DNA. Pertinent screens are usually based on Southern blotting, a time-consuming method that requires large amounts of DNA. Moreover, suitable combinations of restriction enzymes and hybridization sequences may be hard to define or severely limit further options, for example, for cloning of the targeting construct. This ⇑ Corresponding author. Fax: +49 3641 9325932. E-mail address: [email protected] (C. Beetz). 1 These authors contributed equally to this work. 2 Abbreviations used: ESC, embryonic stem cell; PCR, polymerase chain reaction; NEO, neomycin resistance cassette; DTA, diphtheria toxin fragment A; MLPA, multiplex ligation-dependent probe amplification.
is especially critical for knockin approaches that aim to alter specific codons and, therefore, are strongly limited regarding the region that needs to be screened [4]. Polymerase chain reaction (PCR) represents a potential alternative approach but may fail in amplifying long homologous recombination arms or be too unspecific for differentiating between distinct nuclease-mediated alterations. We aimed at generating a knockout of the murine Reep2 gene in order to model hereditary spastic paraplegia type SPG72 [5,6]. To this end, we applied vector construct DPGS00165_a_F10 as commercially available from the Knockout Mouse Project Repository [7]. It consists of a neomycin resistance cassette (NEO; positive selection marker) that replaces exons 2 to 6 of the gene, two neighboring homology arms of more than 4 kb each, and a sequence encoding diphtheria toxin fragment A (DTA; negative selection marker) (Fig. 1B). We had initially planned to screen ESC clones by conventional Southern blotting and sought to design a set of suitable strategies. There was, however, only one reasonable option; the use of EcoRV together with a 50 -located probe was expected to reveal 11.1- and 8.8-kb bands from the wild-type and targeted alleles, respectively (see Supplementary Fig. 1 in online supplementary material). In test experiments, we consistently observed two unspecific bands, one of which was in the size range predicted for the targeted restriction fragment (cf. Fig. 1D). Given the expected difficulties with specificity and sensitivity of the conventional screening strategies, we considered the copy number detection tool multiplex ligation-dependent probe ampli-
http://dx.doi.org/10.1016/j.ab.2015.01.007 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: E. Elert-Dobkowska et al., Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.01.007
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YABIO 11951
No. of Pages 3, Model 5G
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Notes & Tips / E. Elert-Dobkowska et al. / Anal. Biochem. xxx (2015) xxx–xxx
Fig.1. Multiplex ligation-dependent probe amplification (MLPA)-based screening of murine embryonic stem cell (ESC) clones for homologous recombination after electroporation of a Reep2 knockout construct. (A) Schematic outline of MLPA. Pairs of oligonucleotides (‘‘half-probes’’) hybridize to single-stranded genomic DNA. ‘‘Fullprobes,’’ as resulting from ligation (gray spot) at the remaining nick, are amplified by PCR. Genomic deletions (middle) or replacements (right) remove hybridization target compared with unchanged loci (left), thereby reducing the amount of resulting PCR product. (B) To scale scheme of the Reep2 gene and the targeting construct after linearization with AsiSI. Exons are represented as vertical bars, with untranslated regions and coding sequence depicted narrow and wide, respectively. Triangles indicate MLPA probe targeting sites. Thick horizontal line in lower panel marks construct-specific sequence. 3UTR, 30 untranslated region; DTA, diphtheria toxin fragment A-encoding sequence; ex04, exon 4; in02, intron 2; NEO, neomycin resistance cassette; prom, promoter. (C) Summary of MLPA-based screening of neomycin-resistant ESC clones. Relative MLPA signals for 151 clones not showing evidence for reduced Reep2 copy number are summarized as box plots. Values for the five clones considered candidates for successful homologous recombination (see text) are depicted as small circles differing in gray scale. Horizontal stippled lines (at values 0.70 and 1.30) mark the range of signals for a normal copy number. Note that the only signals below 0.70 concern MLPA probes Reep2_in02 and Reep2_ex04 in the five candidate clones (circled by dotted line). Abbreviations are as in panel B. (D) Southern blotting-based confirmation of three positive and of two negative clones (see panel B).
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fication (MLPA; Fig. 1A) [8] as a potential alternative platform. We reasoned that the replaced part of the gene should be present as a single copy in the genome of successfully targeted ESCs, whereas sequence from the homology arms should remain detectable in two copies. In contrast, unspecific integration of the construct would retain the normal two copies for the intended deletion region, whereas the homology arms can be expected to be present in even three copies assuming cointegration with the positive selection marker. With two MLPA probes placed in the deleted region, one probe each targeting the 30 and 50 homology arms (Fig. 1B), and an additional five control probes, our homemade MLPA set consisted of a total of nine probes (corresponding oligonucleotide sequences are provided in Supplementary Fig. 2).
For obtaining the clones to be screened, the construct was linearized with AsiSI and electroporated into R1 mouse ESCs. Genomic DNA from 163 clones was prepared using a standard protocol that involves a proteinase K digest followed by precipitation. Roughly 300 ng of DNA was used for setting up MLPA reactions with reagents from the EK5 kit (MRC-Holland, Amsterdam, The Netherlands). We followed the protocol that is enclosed in this kit and freely available at http://www.MLPA.com. MLPA products were visualized and data were analyzed as described previously [9]. In short, the amounts of DNA amplified in the PCR step of MLPA were estimated by densitometry. They were then normalized within and across samples to finally derive a unit-less relative MLPA signal to which each genomic copy is expected to contribute approximately
Please cite this article in press as: E. Elert-Dobkowska et al., Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.01.007
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12 February 2015 Notes & Tips / E. Elert-Dobkowska et al. / Anal. Biochem. xxx (2015) xxx–xxx 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153
0.5. A total of 20 ESC-derived DNAs from an unrelated project served as reference. For 12 of the Reep2 construct-derived clones, data quality was considered as too low because standard deviations for the control probes exceeded 20%. Analysis of the remaining 151 data sets revealed nearly all relative MLPA signals to be above 0.70, that is, above the cutoff commonly considered to indicate reduced copy number [8]. The exceptions concerned five clones in which both Reep2 probes that target the deleted region showed relative MLPA signals between 0.49 and 0.69 (Fig. 1C). Because these findings were confirmed on independent secondary DNA preparations (not shown), we considered these five clones as excellent candidates for successful targeting by homologous recombination. Of note, we also observed a significant signal increase for the Reep2 30 UTR probe in many of the noncandidates (Fig. 1C). This would be consistent with unspecific cointegration of the positive selection marker NEO and the physically close 30 homology arm, but not the far more distant 50 homology arm (cf. Fig. 1B). In an attempt to corroborate the MLPA findings for the five candidate clones, we tried to amplify a fragment across the homology arms by long-range PCR. When applying nested primers, but not in conventional PCR, products of the expected size were occasionally generated (not shown). Due to low reproducibility, however, we considered long-range PCR as too unreliable. Therefore, we finally turned to our initially developed Southern blotting strategy (see above). Despite close proximity of the previously noted unspecific band, a recombination-specific signal was observed for all five MLPA-positive candidate clones but for none of the MLPA-negative ones (Fig. 1D). Thus, MLPA turned out to be highly specific and sensitive for detecting ESC clones in which our construct had integrated by homologous recombination. The screening strategy presented here for identification of Reep2 recombinants is applicable to any genomic mouse locus targeted by a (partial) deletion construct. Moreover, the high sensitivity of MLPA should also enable detection of smaller alterations. Indeed, a previous study detected a single base exchange as well as a loxP site integration in the murine PCNA gene [10]. MLPA-based screening of ESCs also possesses great potential in combination with the novel nuclease-mediated genome engineering systems [3]. Here, the desired DNA repair needs to be identified against a background of numerous alternative repair options, and accordingly designed MLPA probes should be able to specifically
3
recognize the presence of the intended sequence. In summary, we believe that screening of murine ESCs in the frame of genome editing projects represents another useful addition to the already extensive set of applications of the MLPA technology.
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Acknowledgments
158
This study was supported by a Grant from Gentechnologiestiftung – Dr. Georg und Ingeborg Scheel Stiftung (to C.B.). We thank K. Schorr and K. Stein for excellent technical assistance.
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Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.01.007.
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References
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[1] G.G. Hicks, E.G. Shi, X.M. Li, C.H. Li, M. Pawlak, H.E. Ruley, Functional genomics in mice by tagged sequence mutagenesis, Nat. Genet. 16 (1997) 338–344. [2] K.R. Thomas, M.R. Capecchi, Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells, Cell 51 (1987) 503–512. [3] R.M. Gupta, K. Musunuru, Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9, J. Clin. Invest. 124 (2014) 4154–4161. [4] E. Leipold, L. Liebmann, G.C. Korenke, T. Heinrich, S. Giesselmann, L. Baets, M. Ebbinghaus, R.O. Goral, T. Stödberg, J.C. Hennings, et al., A de novo gain-offunction mutation in SCN11A causes loss of pain perception, Nat. Genet. 45 (2013) 1399–1404. [5] T. Esteves, A. Durr, E. Mundwiller, J.L. Loureiro, M. Boutry, M.A. Gonzalez, J. Gauthier, K.H. El-Hachimi, C. Depienne, M.P. Muriel, et al., Loss of association of REEP2 with membranes leads to hereditary spastic paraplegia, Am. J. Hum. Genet. 94 (2014) 268–277. [6] G. Novarino, A.G. Fenstermaker, M.S. Zaki, M. Hofree, J.L. Silhavy, A.D. Heiberg, M. Abdellateef, B. Rosti, E. Scott, L. Mansour, et al., Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders, Science 343 (2014) 506–511. [7] K.C. Lloyd, A knockout mouse resource for the biomedical research community, Ann. N. Y. Acad. Sci. 1245 (2011) 24–26. [8] J.P. Schouten, C.J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, G. Pals, Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification, Nucleic Acids Res. 30 (2002) e57. [9] C. Beetz, A.O. Nygren, J. Schickel, M. Auer-Grumbach, K. Burk, G. Heide, J. Kassubek, S. Klimpe, T. Klopstock, F. Kreuz, et al., High frequency of partial SPAST deletions in autosomal dominant hereditary spastic paraplegia, Neurology 67 (2006) 1926–1930. [10] P. Langerak, A.O. Nygren, J.P. Schouten, H. Jacobs, Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA, Nucleic Acids Res. 33 (2005) e188.
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Please cite this article in press as: E. Elert-Dobkowska et al., Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.01.007
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No. of Pages 3, Model 5G
12 February 2015 Analytical Biochemistry xxx (2015) xxx–xxx 1
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
2
Notes & Tips
6 4 7 5
Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones
8
Ewelina Elert-Dobkowska a,b,1, J. Christopher Hennings c,1, Christian A. Hübner c, Christian Beetz a,⇑
9 10 11
12 1 2 4 7 15 16 17 18 19 20 21 22 23 24 25 26
a
Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, 07747 Jena, Germany Department of Genetics, Institute of Psychiatry and Neurology, 02-957 Warsaw, Poland c Institute of Human Genetics, Jena University Hospital, 07747 Jena, Germany b
a r t i c l e
i n f o
Article history: Received 27 October 2014 Received in revised form 9 January 2015 Accepted 12 January 2015 Available online xxxx Keywords: Embryonic stem cells Genome engineering Knockout MLPA Mouse model
a b s t r a c t Following locus-specific genome editing of mouse embryonic stem cells (ESCs), the identification of correctly targeted clones remains a challenge. We applied multiplex ligation-dependent probe amplification (MLPA) to screen for homologous recombination-based genomic integration of a knockout construct in which part of a gene is deleted. All candidate ESCs thereby identified were subsequently validated by conventional methods. Thus, MLPA represents a highly reliable as well as cost- and time-efficient alternative to currently applied methods such as Southern blotting and polymerase chain reaction (PCR)-based approaches. It is also applicable to knockin recombination strategies and compatible with the CRISPR/Cas9 system and other genome editing strategies. Ó 2015 Elsevier Inc. All rights reserved.
28 29 30 31 32 33 34 35 36 37
38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59
Genetic engineering of model organisms has opened new opportunities for understanding gene function and thereby revolutionized many research areas. From a biomedical perspective, the editing of the mouse genome has proven to be most powerful. There are two fundamentally differing approaches for genotypedriven mutagenesis of murine embryonic stem cells (ESCs).2 One is the random insertion of a universal sequence cassette into the genome followed by complementary DNA (cDNA)-based identification of the desired intragenic events (‘‘gene trapping’’ [1]). The other is the alteration of predefined loci either by homologous recombination with exogenously provided DNA [2] or by the combination of site-specific nucleases with a DNA-modifying repair process [3]. In these locus-specific methods, a varying proportion of ESC clones obtained does not carry the intended alteration. Identification of correctly targeted clones (often accounting for <1%) requires analysis of their genomic DNA. Pertinent screens are usually based on Southern blotting, a time-consuming method that requires large amounts of DNA. Moreover, suitable combinations of restriction enzymes and hybridization sequences may be hard to define or severely limit further options, for example, for cloning of the targeting construct. This ⇑ Corresponding author. Fax: +49 3641 9325932. E-mail address: [email protected] (C. Beetz). 1 These authors contributed equally to this work. 2 Abbreviations used: ESC, embryonic stem cell; PCR, polymerase chain reaction; NEO, neomycin resistance cassette; DTA, diphtheria toxin fragment A; MLPA, multiplex ligation-dependent probe amplification.
is especially critical for knockin approaches that aim to alter specific codons and, therefore, are strongly limited regarding the region that needs to be screened [4]. Polymerase chain reaction (PCR) represents a potential alternative approach but may fail in amplifying long homologous recombination arms or be too unspecific for differentiating between distinct nuclease-mediated alterations. We aimed at generating a knockout of the murine Reep2 gene in order to model hereditary spastic paraplegia type SPG72 [5,6]. To this end, we applied vector construct DPGS00165_a_F10 as commercially available from the Knockout Mouse Project Repository [7]. It consists of a neomycin resistance cassette (NEO; positive selection marker) that replaces exons 2 to 6 of the gene, two neighboring homology arms of more than 4 kb each, and a sequence encoding diphtheria toxin fragment A (DTA; negative selection marker) (Fig. 1B). We had initially planned to screen ESC clones by conventional Southern blotting and sought to design a set of suitable strategies. There was, however, only one reasonable option; the use of EcoRV together with a 50 -located probe was expected to reveal 11.1- and 8.8-kb bands from the wild-type and targeted alleles, respectively (see Supplementary Fig. 1 in online supplementary material). In test experiments, we consistently observed two unspecific bands, one of which was in the size range predicted for the targeted restriction fragment (cf. Fig. 1D). Given the expected difficulties with specificity and sensitivity of the conventional screening strategies, we considered the copy number detection tool multiplex ligation-dependent probe ampli-
http://dx.doi.org/10.1016/j.ab.2015.01.007 0003-2697/Ó 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: E. Elert-Dobkowska et al., Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.01.007
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No. of Pages 3, Model 5G
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Notes & Tips / E. Elert-Dobkowska et al. / Anal. Biochem. xxx (2015) xxx–xxx
Fig.1. Multiplex ligation-dependent probe amplification (MLPA)-based screening of murine embryonic stem cell (ESC) clones for homologous recombination after electroporation of a Reep2 knockout construct. (A) Schematic outline of MLPA. Pairs of oligonucleotides (‘‘half-probes’’) hybridize to single-stranded genomic DNA. ‘‘Fullprobes,’’ as resulting from ligation (gray spot) at the remaining nick, are amplified by PCR. Genomic deletions (middle) or replacements (right) remove hybridization target compared with unchanged loci (left), thereby reducing the amount of resulting PCR product. (B) To scale scheme of the Reep2 gene and the targeting construct after linearization with AsiSI. Exons are represented as vertical bars, with untranslated regions and coding sequence depicted narrow and wide, respectively. Triangles indicate MLPA probe targeting sites. Thick horizontal line in lower panel marks construct-specific sequence. 3UTR, 30 untranslated region; DTA, diphtheria toxin fragment A-encoding sequence; ex04, exon 4; in02, intron 2; NEO, neomycin resistance cassette; prom, promoter. (C) Summary of MLPA-based screening of neomycin-resistant ESC clones. Relative MLPA signals for 151 clones not showing evidence for reduced Reep2 copy number are summarized as box plots. Values for the five clones considered candidates for successful homologous recombination (see text) are depicted as small circles differing in gray scale. Horizontal stippled lines (at values 0.70 and 1.30) mark the range of signals for a normal copy number. Note that the only signals below 0.70 concern MLPA probes Reep2_in02 and Reep2_ex04 in the five candidate clones (circled by dotted line). Abbreviations are as in panel B. (D) Southern blotting-based confirmation of three positive and of two negative clones (see panel B).
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fication (MLPA; Fig. 1A) [8] as a potential alternative platform. We reasoned that the replaced part of the gene should be present as a single copy in the genome of successfully targeted ESCs, whereas sequence from the homology arms should remain detectable in two copies. In contrast, unspecific integration of the construct would retain the normal two copies for the intended deletion region, whereas the homology arms can be expected to be present in even three copies assuming cointegration with the positive selection marker. With two MLPA probes placed in the deleted region, one probe each targeting the 30 and 50 homology arms (Fig. 1B), and an additional five control probes, our homemade MLPA set consisted of a total of nine probes (corresponding oligonucleotide sequences are provided in Supplementary Fig. 2).
For obtaining the clones to be screened, the construct was linearized with AsiSI and electroporated into R1 mouse ESCs. Genomic DNA from 163 clones was prepared using a standard protocol that involves a proteinase K digest followed by precipitation. Roughly 300 ng of DNA was used for setting up MLPA reactions with reagents from the EK5 kit (MRC-Holland, Amsterdam, The Netherlands). We followed the protocol that is enclosed in this kit and freely available at http://www.MLPA.com. MLPA products were visualized and data were analyzed as described previously [9]. In short, the amounts of DNA amplified in the PCR step of MLPA were estimated by densitometry. They were then normalized within and across samples to finally derive a unit-less relative MLPA signal to which each genomic copy is expected to contribute approximately
Please cite this article in press as: E. Elert-Dobkowska et al., Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.01.007
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12 February 2015 Notes & Tips / E. Elert-Dobkowska et al. / Anal. Biochem. xxx (2015) xxx–xxx 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153
0.5. A total of 20 ESC-derived DNAs from an unrelated project served as reference. For 12 of the Reep2 construct-derived clones, data quality was considered as too low because standard deviations for the control probes exceeded 20%. Analysis of the remaining 151 data sets revealed nearly all relative MLPA signals to be above 0.70, that is, above the cutoff commonly considered to indicate reduced copy number [8]. The exceptions concerned five clones in which both Reep2 probes that target the deleted region showed relative MLPA signals between 0.49 and 0.69 (Fig. 1C). Because these findings were confirmed on independent secondary DNA preparations (not shown), we considered these five clones as excellent candidates for successful targeting by homologous recombination. Of note, we also observed a significant signal increase for the Reep2 30 UTR probe in many of the noncandidates (Fig. 1C). This would be consistent with unspecific cointegration of the positive selection marker NEO and the physically close 30 homology arm, but not the far more distant 50 homology arm (cf. Fig. 1B). In an attempt to corroborate the MLPA findings for the five candidate clones, we tried to amplify a fragment across the homology arms by long-range PCR. When applying nested primers, but not in conventional PCR, products of the expected size were occasionally generated (not shown). Due to low reproducibility, however, we considered long-range PCR as too unreliable. Therefore, we finally turned to our initially developed Southern blotting strategy (see above). Despite close proximity of the previously noted unspecific band, a recombination-specific signal was observed for all five MLPA-positive candidate clones but for none of the MLPA-negative ones (Fig. 1D). Thus, MLPA turned out to be highly specific and sensitive for detecting ESC clones in which our construct had integrated by homologous recombination. The screening strategy presented here for identification of Reep2 recombinants is applicable to any genomic mouse locus targeted by a (partial) deletion construct. Moreover, the high sensitivity of MLPA should also enable detection of smaller alterations. Indeed, a previous study detected a single base exchange as well as a loxP site integration in the murine PCNA gene [10]. MLPA-based screening of ESCs also possesses great potential in combination with the novel nuclease-mediated genome engineering systems [3]. Here, the desired DNA repair needs to be identified against a background of numerous alternative repair options, and accordingly designed MLPA probes should be able to specifically
3
recognize the presence of the intended sequence. In summary, we believe that screening of murine ESCs in the frame of genome editing projects represents another useful addition to the already extensive set of applications of the MLPA technology.
154
Acknowledgments
158
This study was supported by a Grant from Gentechnologiestiftung – Dr. Georg und Ingeborg Scheel Stiftung (to C.B.). We thank K. Schorr and K. Stein for excellent technical assistance.
159
Appendix A. Supplementary data
162
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ab.2015.01.007.
163
References
165
[1] G.G. Hicks, E.G. Shi, X.M. Li, C.H. Li, M. Pawlak, H.E. Ruley, Functional genomics in mice by tagged sequence mutagenesis, Nat. Genet. 16 (1997) 338–344. [2] K.R. Thomas, M.R. Capecchi, Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells, Cell 51 (1987) 503–512. [3] R.M. Gupta, K. Musunuru, Expanding the genetic editing tool kit: ZFNs, TALENs, and CRISPR-Cas9, J. Clin. Invest. 124 (2014) 4154–4161. [4] E. Leipold, L. Liebmann, G.C. Korenke, T. Heinrich, S. Giesselmann, L. Baets, M. Ebbinghaus, R.O. Goral, T. Stödberg, J.C. Hennings, et al., A de novo gain-offunction mutation in SCN11A causes loss of pain perception, Nat. Genet. 45 (2013) 1399–1404. [5] T. Esteves, A. Durr, E. Mundwiller, J.L. Loureiro, M. Boutry, M.A. Gonzalez, J. Gauthier, K.H. El-Hachimi, C. Depienne, M.P. Muriel, et al., Loss of association of REEP2 with membranes leads to hereditary spastic paraplegia, Am. J. Hum. Genet. 94 (2014) 268–277. [6] G. Novarino, A.G. Fenstermaker, M.S. Zaki, M. Hofree, J.L. Silhavy, A.D. Heiberg, M. Abdellateef, B. Rosti, E. Scott, L. Mansour, et al., Exome sequencing links corticospinal motor neuron disease to common neurodegenerative disorders, Science 343 (2014) 506–511. [7] K.C. Lloyd, A knockout mouse resource for the biomedical research community, Ann. N. Y. Acad. Sci. 1245 (2011) 24–26. [8] J.P. Schouten, C.J. McElgunn, R. Waaijer, D. Zwijnenburg, F. Diepvens, G. Pals, Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification, Nucleic Acids Res. 30 (2002) e57. [9] C. Beetz, A.O. Nygren, J. Schickel, M. Auer-Grumbach, K. Burk, G. Heide, J. Kassubek, S. Klimpe, T. Klopstock, F. Kreuz, et al., High frequency of partial SPAST deletions in autosomal dominant hereditary spastic paraplegia, Neurology 67 (2006) 1926–1930. [10] P. Langerak, A.O. Nygren, J.P. Schouten, H. Jacobs, Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA, Nucleic Acids Res. 33 (2005) e188.
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Please cite this article in press as: E. Elert-Dobkowska et al., Multiplex ligation-dependent probe amplification for identification of correctly targeted murine embryonic stem cell clones, Anal. Biochem. (2015), http://dx.doi.org/10.1016/j.ab.2015.01.007
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