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One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

Ohtsuka M, Miura H, Mochida K, Hirose M, Hasegawa A, Ogura A, Mizutani R, Kimura M, Isotani A, Ikawa M, Sato M, Gurumurthy CB - BMC Genomics (2015)

Bottom Line: The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported.The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background.Further, the i-PITT system is freely accessible to scientific community.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.

ABSTRACT

Background: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

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PCR-based genotyping. (A) The various possible allele outcomes and primer sets (a - i) used for detecting them. The black and blue arrows indicate universal and DOI-specific primers, respectively. The green arrows indicate semi-universal primers used for identification of targeted insertion for several constructs including pBGX and pBGT. (B) Example of PCR-based genotyping. PCR was performed using 12 samples derived from mixed injection of pBGX and pBGT (see Figure 4E). The primer sets used were as follows (from the top to the bottom): “M273/M839”, “M273/M874”, “M274/M376”, “M645/M646”, “M873/M874”, “#235/M026”, “M273/M879”, “M958/M839”, “M958/M874”, “M953/M839”, “M953/M874”, “M953/M026” and “M273/M026”. See text for more details.
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Fig5: PCR-based genotyping. (A) The various possible allele outcomes and primer sets (a - i) used for detecting them. The black and blue arrows indicate universal and DOI-specific primers, respectively. The green arrows indicate semi-universal primers used for identification of targeted insertion for several constructs including pBGX and pBGT. (B) Example of PCR-based genotyping. PCR was performed using 12 samples derived from mixed injection of pBGX and pBGT (see Figure 4E). The primer sets used were as follows (from the top to the bottom): “M273/M839”, “M273/M874”, “M274/M376”, “M645/M646”, “M873/M874”, “#235/M026”, “M273/M879”, “M958/M839”, “M958/M874”, “M953/M839”, “M953/M874”, “M953/M026” and “M273/M026”. See text for more details.

Mentions: Theoretically, co-introduction of both iCre and PhiC31o mRNAs can result in one of several possible insertion alleles such as TIex allele 1, 2, 4, 5 and 6 (Figure 1, Additional file 1: Figure S3 and S4). TIex allele 1 and 2 are the result of Cre-based recombination or PhiC31-based integration systems, respectively. Various genotyping assays were employed to detect targeted insertion of DOI: primer sets ‘a’, ‘b’, ‘d’ and ‘e’ amplify the 5’ or 3’ junction regions generated by recombination/integration whereas primer set ‘c’ amplifies internal regions of the transgene (Figure 5A). Targeted insertion of DOI generated by Cre-based recombination only (TIex allele 1) or by PhiC31 integration system only (TIex allele 2) can be detected with the primer sets ‘a’ and ‘b’ (for Cre-mediated recombination) or primer sets ‘a’ and ‘d’ (for PhiC31-mediated integration). Since PCR with primer set ‘a’ is applicable to identification of targeted insertion of DOI performed by either systems, we used this primer set for the first screening in all i-PITT experiments.Figure 5


One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT).

Ohtsuka M, Miura H, Mochida K, Hirose M, Hasegawa A, Ogura A, Mizutani R, Kimura M, Isotani A, Ikawa M, Sato M, Gurumurthy CB - BMC Genomics (2015)

PCR-based genotyping. (A) The various possible allele outcomes and primer sets (a - i) used for detecting them. The black and blue arrows indicate universal and DOI-specific primers, respectively. The green arrows indicate semi-universal primers used for identification of targeted insertion for several constructs including pBGX and pBGT. (B) Example of PCR-based genotyping. PCR was performed using 12 samples derived from mixed injection of pBGX and pBGT (see Figure 4E). The primer sets used were as follows (from the top to the bottom): “M273/M839”, “M273/M874”, “M274/M376”, “M645/M646”, “M873/M874”, “#235/M026”, “M273/M879”, “M958/M839”, “M958/M874”, “M953/M839”, “M953/M874”, “M953/M026” and “M273/M026”. See text for more details.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4404087&req=5

Fig5: PCR-based genotyping. (A) The various possible allele outcomes and primer sets (a - i) used for detecting them. The black and blue arrows indicate universal and DOI-specific primers, respectively. The green arrows indicate semi-universal primers used for identification of targeted insertion for several constructs including pBGX and pBGT. (B) Example of PCR-based genotyping. PCR was performed using 12 samples derived from mixed injection of pBGX and pBGT (see Figure 4E). The primer sets used were as follows (from the top to the bottom): “M273/M839”, “M273/M874”, “M274/M376”, “M645/M646”, “M873/M874”, “#235/M026”, “M273/M879”, “M958/M839”, “M958/M874”, “M953/M839”, “M953/M874”, “M953/M026” and “M273/M026”. See text for more details.
Mentions: Theoretically, co-introduction of both iCre and PhiC31o mRNAs can result in one of several possible insertion alleles such as TIex allele 1, 2, 4, 5 and 6 (Figure 1, Additional file 1: Figure S3 and S4). TIex allele 1 and 2 are the result of Cre-based recombination or PhiC31-based integration systems, respectively. Various genotyping assays were employed to detect targeted insertion of DOI: primer sets ‘a’, ‘b’, ‘d’ and ‘e’ amplify the 5’ or 3’ junction regions generated by recombination/integration whereas primer set ‘c’ amplifies internal regions of the transgene (Figure 5A). Targeted insertion of DOI generated by Cre-based recombination only (TIex allele 1) or by PhiC31 integration system only (TIex allele 2) can be detected with the primer sets ‘a’ and ‘b’ (for Cre-mediated recombination) or primer sets ‘a’ and ‘d’ (for PhiC31-mediated integration). Since PCR with primer set ‘a’ is applicable to identification of targeted insertion of DOI performed by either systems, we used this primer set for the first screening in all i-PITT experiments.Figure 5

Bottom Line: The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported.The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background.Further, the i-PITT system is freely accessible to scientific community.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Life Science, Division of Basic Medical Science and Molecular Medicine, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan. masato@is.icc.u-tokai.ac.jp.

ABSTRACT

Background: The pronuclear injection (PI) is the simplest and widely used method to generate transgenic (Tg) mice. Unfortunately, PI-based Tg mice show uncertain transgene expression due to random transgene insertion in the genome, usually with multiple copies. Thus, typically at least three or more Tg lines are produced by injecting over 200 zygotes and the best line/s among them are selected through laborious screening steps. Recently, we developed technologies using Cre-loxP system that allow targeted insertion of single-copy transgene into a predetermined locus through PI. We termed the method as PI-based Targeted Transgenesis (PITT). A similar method using PhiC31-attP/B system was reported subsequently.

Results: Here, we developed an improved-PITT (i-PITT) method by combining Cre-loxP, PhiC31-attP/B and FLP-FRT systems directly under C57BL/6N inbred strain, unlike the mixed strain used in previous reports. The targeted Tg efficiency in the i-PITT typically ranged from 10 to 30%, with 47 and 62% in two of the sessions, which is by-far the best Tg rate reported. Furthermore, the system could generate multiple Tg mice simultaneously. We demonstrate that injection of up to three different Tg cassettes in a single injection session into as less as 181 zygotes resulted in production of all three separate Tg DNA containing targeted Tg mice.

Conclusions: The i-PITT system offers several advantages compared to previous methods: multiplexing capability (i-PITT is the only targeted-transgenic method that is proven to generate multiple different transgenic lines simultaneously), very high efficiency of targeted-transgenesis (up to 62%), significantly reduces animal numbers in mouse-transgenesis and the system is developed under C57BL/6N strain, the most commonly used pure genetic background. Further, the i-PITT system is freely accessible to scientific community.

Show MeSH
Related in: MedlinePlus