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Efficient detection, quantification and enrichment of subtle allelic alterations.

Chen J, Zhang X, Wang T, Li Z, Guan G, Hong Y - DNA Res. (2012)

Bottom Line: Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing.Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations.Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.

ABSTRACT
Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing. Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations. Efficient detection of such minor alterations remains one of the challenges in ZFN-mediated GT experiments. Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations. In a biallelic model, polyacrylamide gel electrophoresis (PAGE) is capable of detecting rare allelic variations in the form of DNA heteroduplexes at a high efficiency of ~0.4% compared with ~6.3% by the traditional T7 endonuclease I-digestion and agarose gel electrophoresis. In a multiple allelic model, PAGE could discriminate different alleles bearing addition or deletion of 1-18 bp as distinct bands that were easily quantifiable by densitometry. Furthermore, PAGE enables enrichment for rare alleles. We show for the first time that direct endogenous GT is possible in medaka by ZFN RNA injection, whereas PAGE allows for detection and cloning of ZFN-targeted alleles in adults arising from ZFN-injected medaka embryos. Therefore, PAGE is effective for detection, quantification and enrichment of multiple fine allelic differences and thus offers a versatile tool for screening targeted subtle gene alterations.

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Detection of ZFN-mediated allelic alterations in medaka. Medaka embryos were microinjected with RNAs for a pair of ZFNs that targeted the first exon of gsdf locus, and were allowed to develop into adult animals. Genomic DNA was extracted from fin clips of eight randomly sampled adults and was subjected to PCR and PAGE detection of targeted allelic alterations. (A) PAGE profiles of PCR products, unambiguously showing the presence of heteroduplex DNA (asterisks) in two out of eight fish. (B) TAGE profile of PCR products, showing a faint band of heteroduplex DNA (asterisks) within smears in the same two fish.
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DSS023F4: Detection of ZFN-mediated allelic alterations in medaka. Medaka embryos were microinjected with RNAs for a pair of ZFNs that targeted the first exon of gsdf locus, and were allowed to develop into adult animals. Genomic DNA was extracted from fin clips of eight randomly sampled adults and was subjected to PCR and PAGE detection of targeted allelic alterations. (A) PAGE profiles of PCR products, unambiguously showing the presence of heteroduplex DNA (asterisks) in two out of eight fish. (B) TAGE profile of PCR products, showing a faint band of heteroduplex DNA (asterisks) within smears in the same two fish.

Mentions: We are interested in the development of approaches for direct GT by using ZFNs and TALENs in medaka. By microinjection of RNAs for a pair ZFNs into embryos of af medaka, we obtained several targeted alleles at the medaka gsdf, a gene whose RNA expression is spatially and temporally correlated with early testicular differentiation.41 Four of the alleles were used in the multi-allelic model assay described above (Figs 2 and 3). More than hundred of fish derived from ZFN-injected embryos were grown into adulthood. Genomic DNA was extracted from fin clips of a subset of samples and subjected to TAGE and PAGE assays. We detected successful GT in 2 out of 48 fish examined, because the two fish (4 and 6) exhibited clear bands of Ht PCR products on PAGE (Fig. 4A), and a smear or faint band indicative of Ht products on TAGE (Fig. 4B). The band patterns of two fish samples on PAGE and TAGE were similar to that observed in model systems (Figs 1 and 2). Sequencing 72 recombinant colonies of the Ht PCR products from the third-round PCR (a total of two rounds of grsPCR) revealed three different alleles. One is the WT allele that was present in 45 clones (63%), and the remainder is targeted alleles D7 (22%) and A3D7 (15%; Supplementary Fig. S5). Interestingly, allele A3D7 has both a 3-bp addition and a 7-bp deletion, and more intriguingly, also four mismatches of 1–2 bp each at three separate positions. It follows that ZFNs can simultaneously introduce addition, deletion and mutation into one and same targeted allele. Hence, ZFN-mediated endogenous GT is possible in medaka embryos, and PAGE is effective in detecting and cloning ZFN-targeted rare alleles in fish.Figure 4.


Efficient detection, quantification and enrichment of subtle allelic alterations.

Chen J, Zhang X, Wang T, Li Z, Guan G, Hong Y - DNA Res. (2012)

Detection of ZFN-mediated allelic alterations in medaka. Medaka embryos were microinjected with RNAs for a pair of ZFNs that targeted the first exon of gsdf locus, and were allowed to develop into adult animals. Genomic DNA was extracted from fin clips of eight randomly sampled adults and was subjected to PCR and PAGE detection of targeted allelic alterations. (A) PAGE profiles of PCR products, unambiguously showing the presence of heteroduplex DNA (asterisks) in two out of eight fish. (B) TAGE profile of PCR products, showing a faint band of heteroduplex DNA (asterisks) within smears in the same two fish.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3473374&req=5

DSS023F4: Detection of ZFN-mediated allelic alterations in medaka. Medaka embryos were microinjected with RNAs for a pair of ZFNs that targeted the first exon of gsdf locus, and were allowed to develop into adult animals. Genomic DNA was extracted from fin clips of eight randomly sampled adults and was subjected to PCR and PAGE detection of targeted allelic alterations. (A) PAGE profiles of PCR products, unambiguously showing the presence of heteroduplex DNA (asterisks) in two out of eight fish. (B) TAGE profile of PCR products, showing a faint band of heteroduplex DNA (asterisks) within smears in the same two fish.
Mentions: We are interested in the development of approaches for direct GT by using ZFNs and TALENs in medaka. By microinjection of RNAs for a pair ZFNs into embryos of af medaka, we obtained several targeted alleles at the medaka gsdf, a gene whose RNA expression is spatially and temporally correlated with early testicular differentiation.41 Four of the alleles were used in the multi-allelic model assay described above (Figs 2 and 3). More than hundred of fish derived from ZFN-injected embryos were grown into adulthood. Genomic DNA was extracted from fin clips of a subset of samples and subjected to TAGE and PAGE assays. We detected successful GT in 2 out of 48 fish examined, because the two fish (4 and 6) exhibited clear bands of Ht PCR products on PAGE (Fig. 4A), and a smear or faint band indicative of Ht products on TAGE (Fig. 4B). The band patterns of two fish samples on PAGE and TAGE were similar to that observed in model systems (Figs 1 and 2). Sequencing 72 recombinant colonies of the Ht PCR products from the third-round PCR (a total of two rounds of grsPCR) revealed three different alleles. One is the WT allele that was present in 45 clones (63%), and the remainder is targeted alleles D7 (22%) and A3D7 (15%; Supplementary Fig. S5). Interestingly, allele A3D7 has both a 3-bp addition and a 7-bp deletion, and more intriguingly, also four mismatches of 1–2 bp each at three separate positions. It follows that ZFNs can simultaneously introduce addition, deletion and mutation into one and same targeted allele. Hence, ZFN-mediated endogenous GT is possible in medaka embryos, and PAGE is effective in detecting and cloning ZFN-targeted rare alleles in fish.Figure 4.

Bottom Line: Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing.Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations.Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.

ABSTRACT
Gene targeting (GT) can introduce subtle alterations into a particular locus and represents a powerful tool for genome editing. Engineered zinc finger nucleases (ZFNs) are effective for generating minor allelic alterations. Efficient detection of such minor alterations remains one of the challenges in ZFN-mediated GT experiments. Here, we report the establishment of procedures allowing for efficient detection, quantification and enrichment of such subtle alterations. In a biallelic model, polyacrylamide gel electrophoresis (PAGE) is capable of detecting rare allelic variations in the form of DNA heteroduplexes at a high efficiency of ~0.4% compared with ~6.3% by the traditional T7 endonuclease I-digestion and agarose gel electrophoresis. In a multiple allelic model, PAGE could discriminate different alleles bearing addition or deletion of 1-18 bp as distinct bands that were easily quantifiable by densitometry. Furthermore, PAGE enables enrichment for rare alleles. We show for the first time that direct endogenous GT is possible in medaka by ZFN RNA injection, whereas PAGE allows for detection and cloning of ZFN-targeted alleles in adults arising from ZFN-injected medaka embryos. Therefore, PAGE is effective for detection, quantification and enrichment of multiple fine allelic differences and thus offers a versatile tool for screening targeted subtle gene alterations.

Show MeSH
Related in: MedlinePlus