The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats
Determination of the genomic copy number of Cre transgene is critical for the establishment of transgenic animal models. We demonstrate a real-time quantitative polymerase chain reaction (RT-qPCR) based method for genomic copy number determination of improved Cre (iCre), which was tested in a transg...
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| Language: | English |
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Taylor & Francis Group
2024-12-01
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| Series: | All Life |
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| Online Access: | http://dx.doi.org/10.1080/26895293.2024.2316078 |
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| author | Ge Diao Min Tian Runbo Li Jie Huang Jian Han Jianxin Guo |
| author_facet | Ge Diao Min Tian Runbo Li Jie Huang Jian Han Jianxin Guo |
| author_sort | Ge Diao |
| collection | DOAJ |
| description | Determination of the genomic copy number of Cre transgene is critical for the establishment of transgenic animal models. We demonstrate a real-time quantitative polymerase chain reaction (RT-qPCR) based method for genomic copy number determination of improved Cre (iCre), which was tested in a transgenic mouse model of CYP19A1-iCre. The standards were derived from tandem repeats of iCre fragments which were constructed by a new application of megaprimer PCR. Two rounds of megaprimer PCR were used to obtain specific tandem repeats of iCre fragments, which were subsequently cloned into pUCm-T vectors. After the fragments of IL-2 (reference gene) were ligated into the same vectors, standards with copy number ratios (CNR) of 0.5, 1, 2, 4, 8, and 16 between iCre and IL-2 were completed. The R2 values of the standard curves exceeded 0.99. The calculated CNR value of the quality control sample was satisfactory. Multiple copies of iCre were detected in founder mice. After several passages, the copy number of iCre gradually decreased to a single copy, which was consistent with theoretical expectation. The established method is a universal and sensitive program for genomic copy number determination of iCre in transgenic mice. |
| format | Article |
| id | doaj-art-60dccc766b2d42349721418f13dcbb07 |
| institution | Kabale University |
| issn | 2689-5307 |
| language | English |
| publishDate | 2024-12-01 |
| publisher | Taylor & Francis Group |
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| series | All Life |
| spelling | doaj-art-60dccc766b2d42349721418f13dcbb072025-01-20T14:38:00ZengTaylor & Francis GroupAll Life2689-53072024-12-01170110.1080/26895293.2024.23160782316078The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeatsGe Diao0Min Tian1Runbo Li2Jie Huang3Jian Han4Jianxin Guo5Army Medical University (Third Military Medical University)Army Medical University (Third Military Medical University)Army Medical University (Third Military Medical University)Army Medical University (Third Military Medical University)Army Medical University (Third Military Medical University)Army Medical University (Third Military Medical University)Determination of the genomic copy number of Cre transgene is critical for the establishment of transgenic animal models. We demonstrate a real-time quantitative polymerase chain reaction (RT-qPCR) based method for genomic copy number determination of improved Cre (iCre), which was tested in a transgenic mouse model of CYP19A1-iCre. The standards were derived from tandem repeats of iCre fragments which were constructed by a new application of megaprimer PCR. Two rounds of megaprimer PCR were used to obtain specific tandem repeats of iCre fragments, which were subsequently cloned into pUCm-T vectors. After the fragments of IL-2 (reference gene) were ligated into the same vectors, standards with copy number ratios (CNR) of 0.5, 1, 2, 4, 8, and 16 between iCre and IL-2 were completed. The R2 values of the standard curves exceeded 0.99. The calculated CNR value of the quality control sample was satisfactory. Multiple copies of iCre were detected in founder mice. After several passages, the copy number of iCre gradually decreased to a single copy, which was consistent with theoretical expectation. The established method is a universal and sensitive program for genomic copy number determination of iCre in transgenic mice.http://dx.doi.org/10.1080/26895293.2024.2316078megaprimer pcrtandem repeatcre transgenegenomic copy numberreal-time quantitative pcr |
| spellingShingle | Ge Diao Min Tian Runbo Li Jie Huang Jian Han Jianxin Guo The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats All Life megaprimer pcr tandem repeat cre transgene genomic copy number real-time quantitative pcr |
| title | The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats |
| title_full | The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats |
| title_fullStr | The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats |
| title_full_unstemmed | The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats |
| title_short | The genomic copy number determination of improved Cre in transgenic mice using standards derived from megaprimer PCR constructed tandem repeats |
| title_sort | genomic copy number determination of improved cre in transgenic mice using standards derived from megaprimer pcr constructed tandem repeats |
| topic | megaprimer pcr tandem repeat cre transgene genomic copy number real-time quantitative pcr |
| url | http://dx.doi.org/10.1080/26895293.2024.2316078 |
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