Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction
Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes. As pa...
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| Veröffentlicht in: | BMC cell biology 2016-08, Vol.17 (1), p.30-30, Article 30 |
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| creator | Codner, Gemma F Lindner, Loic Caulder, Adam Wattenhofer-Donzé, Marie Radage, Adam Mertz, Annelyse Eisenmann, Benjamin Mianné, Joffrey Evans, Edward P Beechey, Colin V Fray, Martin D Birling, Marie-Christine Hérault, Yann Pavlovic, Guillaume Teboul, Lydia |
| description | Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes.
As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost.
We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV). |
| doi_str_mv | 10.1186/s12860-016-0108-6 |
| format | Article |
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As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost.
We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).</description><identifier>ISSN: 1471-2121</identifier><identifier>EISSN: 1471-2121</identifier><identifier>DOI: 10.1186/s12860-016-0108-6</identifier><identifier>PMID: 27496052</identifier><language>eng</language><publisher>England: BioMed Central Ltd</publisher><subject>Aneuploidy ; Animal biology ; Animals ; Cells, Cultured ; Chromosomes, Mammalian - metabolism ; Cloning ; DNA Copy Number Variations ; Embryonic stem cells ; Genetic aspects ; Germ Cells ; Karyotyping - methods ; Life Sciences ; Metaphase ; Methodology ; Mice ; Mice, Inbred C57BL ; Mouse Embryonic Stem Cells - cytology ; Mouse Embryonic Stem Cells - metabolism ; Observations ; Polymerase chain reaction ; Polymerase Chain Reaction - methods</subject><ispartof>BMC cell biology, 2016-08, Vol.17 (1), p.30-30, Article 30</ispartof><rights>COPYRIGHT 2016 BioMed Central Ltd.</rights><rights>Copyright BioMed Central 2016</rights><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><rights>The Author(s). 2016</rights><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c664t-db6c36866909e0ee7d843135564e6cd01fefc86334c5ff4c40776c95fa05e34c3</citedby><cites>FETCH-LOGICAL-c664t-db6c36866909e0ee7d843135564e6cd01fefc86334c5ff4c40776c95fa05e34c3</cites><orcidid>0000-0002-2789-8637 ; 0000-0001-7049-6900</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974727/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4974727/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27496052$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-03680437$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Codner, Gemma F</creatorcontrib><creatorcontrib>Lindner, Loic</creatorcontrib><creatorcontrib>Caulder, Adam</creatorcontrib><creatorcontrib>Wattenhofer-Donzé, Marie</creatorcontrib><creatorcontrib>Radage, Adam</creatorcontrib><creatorcontrib>Mertz, Annelyse</creatorcontrib><creatorcontrib>Eisenmann, Benjamin</creatorcontrib><creatorcontrib>Mianné, Joffrey</creatorcontrib><creatorcontrib>Evans, Edward P</creatorcontrib><creatorcontrib>Beechey, Colin V</creatorcontrib><creatorcontrib>Fray, Martin D</creatorcontrib><creatorcontrib>Birling, Marie-Christine</creatorcontrib><creatorcontrib>Hérault, Yann</creatorcontrib><creatorcontrib>Pavlovic, Guillaume</creatorcontrib><creatorcontrib>Teboul, Lydia</creatorcontrib><title>Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction</title><title>BMC cell biology</title><addtitle>BMC Cell Biol</addtitle><description>Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes.
As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost.
We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).</description><subject>Aneuploidy</subject><subject>Animal biology</subject><subject>Animals</subject><subject>Cells, Cultured</subject><subject>Chromosomes, Mammalian - metabolism</subject><subject>Cloning</subject><subject>DNA Copy Number Variations</subject><subject>Embryonic stem cells</subject><subject>Genetic aspects</subject><subject>Germ Cells</subject><subject>Karyotyping - methods</subject><subject>Life Sciences</subject><subject>Metaphase</subject><subject>Methodology</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Mouse Embryonic Stem Cells - cytology</subject><subject>Mouse Embryonic Stem Cells - metabolism</subject><subject>Observations</subject><subject>Polymerase chain reaction</subject><subject>Polymerase Chain Reaction - methods</subject><issn>1471-2121</issn><issn>1471-2121</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNptkl2L3CAUhkNp6X60P6A3RehN9yJbjUaTm8KwtN3CQG_aa3HMccat0VSThfz7KrMddpciohyf93g8vlX1juBrQjr-KZGm47jGhOeJu5q_qM4JE6RuSENePtqfVRcp3WFMREfw6-qsEaznuG3Oq3XjYZlcsMOKko4A3vo9CgbBuItr8FajNMOINDiHtAseEtqtaIRZTQeVAP1WGZvXqciUH9AQw-RgRoPd21k5NAW3jhALqg_KehRB6dkG_6Z6ZZRL8PZhvax-ff3y8-a23v749v1ms60152yuhx3XlHec97gHDCCGjlFC25Yz4HrAxIDRHaeU6dYYphkWguu-NQq3kIP0svp8zDstuxEGDX6Oyskp2jGXLoOy8umJtwe5D_eS9YKJRuQEV8cEh2ey281WlhjO9WFGxT3J7MeHy2L4s0Ca5WhT6Z3yEJYkSe4_bRnrC_rhGXoXluhzKwpVnksye6L2yoG03oRcoy5J5YbxriOsEzRT1_-h8hhgtDp_mrE5_kRAjgIdQ0oRzOlhBMviLXn0lszeksVbkmfN-8eNPCn-mYn-BTvMyks</recordid><startdate>20160805</startdate><enddate>20160805</enddate><creator>Codner, Gemma F</creator><creator>Lindner, Loic</creator><creator>Caulder, Adam</creator><creator>Wattenhofer-Donzé, Marie</creator><creator>Radage, Adam</creator><creator>Mertz, Annelyse</creator><creator>Eisenmann, Benjamin</creator><creator>Mianné, Joffrey</creator><creator>Evans, Edward P</creator><creator>Beechey, Colin V</creator><creator>Fray, Martin D</creator><creator>Birling, Marie-Christine</creator><creator>Hérault, Yann</creator><creator>Pavlovic, Guillaume</creator><creator>Teboul, Lydia</creator><general>BioMed Central Ltd</general><general>BioMed Central</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7TM</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>7X8</scope><scope>1XC</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0002-2789-8637</orcidid><orcidid>https://orcid.org/0000-0001-7049-6900</orcidid></search><sort><creationdate>20160805</creationdate><title>Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction</title><author>Codner, Gemma F ; Lindner, Loic ; Caulder, Adam ; Wattenhofer-Donzé, Marie ; Radage, Adam ; Mertz, Annelyse ; Eisenmann, Benjamin ; Mianné, Joffrey ; Evans, Edward P ; Beechey, Colin V ; Fray, Martin D ; Birling, Marie-Christine ; Hérault, Yann ; Pavlovic, Guillaume ; Teboul, Lydia</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c664t-db6c36866909e0ee7d843135564e6cd01fefc86334c5ff4c40776c95fa05e34c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>Aneuploidy</topic><topic>Animal biology</topic><topic>Animals</topic><topic>Cells, Cultured</topic><topic>Chromosomes, Mammalian - metabolism</topic><topic>Cloning</topic><topic>DNA Copy Number Variations</topic><topic>Embryonic stem cells</topic><topic>Genetic aspects</topic><topic>Germ Cells</topic><topic>Karyotyping - methods</topic><topic>Life Sciences</topic><topic>Metaphase</topic><topic>Methodology</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Mouse Embryonic Stem Cells - cytology</topic><topic>Mouse Embryonic Stem Cells - metabolism</topic><topic>Observations</topic><topic>Polymerase chain reaction</topic><topic>Polymerase Chain Reaction - methods</topic><toplevel>online_resources</toplevel><creatorcontrib>Codner, Gemma F</creatorcontrib><creatorcontrib>Lindner, Loic</creatorcontrib><creatorcontrib>Caulder, Adam</creatorcontrib><creatorcontrib>Wattenhofer-Donzé, Marie</creatorcontrib><creatorcontrib>Radage, Adam</creatorcontrib><creatorcontrib>Mertz, Annelyse</creatorcontrib><creatorcontrib>Eisenmann, Benjamin</creatorcontrib><creatorcontrib>Mianné, Joffrey</creatorcontrib><creatorcontrib>Evans, Edward P</creatorcontrib><creatorcontrib>Beechey, Colin V</creatorcontrib><creatorcontrib>Fray, Martin D</creatorcontrib><creatorcontrib>Birling, Marie-Christine</creatorcontrib><creatorcontrib>Hérault, Yann</creatorcontrib><creatorcontrib>Pavlovic, Guillaume</creatorcontrib><creatorcontrib>Teboul, Lydia</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Nucleic Acids Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Access via ProQuest (Open Access)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>MEDLINE - Academic</collection><collection>Hyper Article en Ligne (HAL)</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>BMC cell biology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Codner, Gemma F</au><au>Lindner, Loic</au><au>Caulder, Adam</au><au>Wattenhofer-Donzé, Marie</au><au>Radage, Adam</au><au>Mertz, Annelyse</au><au>Eisenmann, Benjamin</au><au>Mianné, Joffrey</au><au>Evans, Edward P</au><au>Beechey, Colin V</au><au>Fray, Martin D</au><au>Birling, Marie-Christine</au><au>Hérault, Yann</au><au>Pavlovic, Guillaume</au><au>Teboul, Lydia</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction</atitle><jtitle>BMC cell biology</jtitle><addtitle>BMC Cell Biol</addtitle><date>2016-08-05</date><risdate>2016</risdate><volume>17</volume><issue>1</issue><spage>30</spage><epage>30</epage><pages>30-30</pages><artnum>30</artnum><issn>1471-2121</issn><eissn>1471-2121</eissn><abstract>Karyotypic integrity is essential for the successful germline transmission of alleles mutated in embryonic stem (ES) cells. Classical methods for the identification of aneuploidy involve cytological analyses that are both time consuming and require rare expertise to identify mouse chromosomes.
As part of the International Mouse Phenotyping Consortium, we gathered data from over 1,500 ES cell clones and found that the germline transmission (GLT) efficiency of clones is compromised when over 50 % of cells harbour chromosome number abnormalities. In JM8 cells, chromosomes 1, 8, 11 or Y displayed copy number variation most frequently, whilst the remainder generally remain unchanged. We developed protocols employing droplet digital polymerase chain reaction (ddPCR) to accurately quantify the copy number of these four chromosomes, allowing efficient triage of ES clones prior to microinjection. We verified that assessments of aneuploidy, and thus decisions regarding the suitability of clones for microinjection, were concordant between classical cytological and ddPCR-based methods. Finally, we improved the method to include assay multiplexing so that two unstable chromosomes are counted simultaneously (and independently) in one reaction, to enhance throughput and further reduce the cost.
We validated a PCR-based method as an alternative to classical karyotype analysis. This technique enables laboratories that are non-specialist, or work with large numbers of clones, to precisely screen ES cells for the most common aneuploidies prior to microinjection to ensure the highest level of germline transmission potential. The application of this method allows early exclusion of aneuploid ES cell clones in the ES cell to mouse conversion process, thus improving the chances of obtaining germline transmission and reducing the number of animals used in failed microinjection attempts. This method can be applied to any other experiments that require accurate analysis of the genome for copy number variation (CNV).</abstract><cop>England</cop><pub>BioMed Central Ltd</pub><pmid>27496052</pmid><doi>10.1186/s12860-016-0108-6</doi><tpages>1</tpages><orcidid>https://orcid.org/0000-0002-2789-8637</orcidid><orcidid>https://orcid.org/0000-0001-7049-6900</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Aneuploidy Animal biology Animals Cells, Cultured Chromosomes, Mammalian - metabolism Cloning DNA Copy Number Variations Embryonic stem cells Genetic aspects Germ Cells Karyotyping - methods Life Sciences Metaphase Methodology Mice Mice, Inbred C57BL Mouse Embryonic Stem Cells - cytology Mouse Embryonic Stem Cells - metabolism Observations Polymerase chain reaction Polymerase Chain Reaction - methods |
| title | Aneuploidy screening of embryonic stem cell clones by metaphase karyotyping and droplet digital polymerase chain reaction |
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