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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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Schematic of PITT-mediated targeted insertion using the new seed mice, TOKOM-3. Cre-PITT (via mutant loxP sites) and PhiC31-PITT (via attB/P site) confer insertion of the donor vector containing. DNA Of Interest (DOI), into a target site in the genome, to generate TIex allele 1 or 2 respectively. DOI shown in this example is tdTomato-poly(A) cassette in this figure. Introduction of FLP removes extra sequence in the TIex allele 1 and 4, resulting in generation of TIΔex allele. See Additional file 1: Figures S1, S3 and S4 for other possible outcomes recombination. GTNOP: a cassette containing “eGFP-T2A-Neomycin resistant gene-hOCT4-PolyA”.
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Fig1: Schematic of PITT-mediated targeted insertion using the new seed mice, TOKOM-3. Cre-PITT (via mutant loxP sites) and PhiC31-PITT (via attB/P site) confer insertion of the donor vector containing. DNA Of Interest (DOI), into a target site in the genome, to generate TIex allele 1 or 2 respectively. DOI shown in this example is tdTomato-poly(A) cassette in this figure. Introduction of FLP removes extra sequence in the TIex allele 1 and 4, resulting in generation of TIΔex allele. See Additional file 1: Figures S1, S3 and S4 for other possible outcomes recombination. GTNOP: a cassette containing “eGFP-T2A-Neomycin resistant gene-hOCT4-PolyA”.

Mentions: The overall scheme of i-PITT is illustrated in Figure 1. A new PITT seed mouse (TOKMO-3) under C57BL/6N genetic background was engineered by inserting a landing pad containing loxP derivatives (JT15 and lox2272), attP, and FRT derivatives (F14, F15 and FRT-L) into the Rosa26 locus through ES cell-based targeting. A donor vector carrying tdTomato-pA, loxP derivatives (JTZ17 and lox2272), attB, and FRT derivatives (F14, F15 and FRT-R) was designed to perform site-specific insertion of a transgene (e.g. “tdTomato-pA” in Figure 1). The transgene flanked by the F14 and F15 sites in the donor vector was inserted into target locus of fertilized eggs via i-PITT (Figure 1 and Additional file 1: Figure S1) that offers options for any of the three enzymes Cre, PhiC31 or FLP to choose for targeted insertion. The resulting embryos will exhibit “targeted insertion with extra sequence (TIex)” allele. The strategy is designed in such way that the extra sequences will be flanked by F14 and F15 elements which enable the removal of extra sequences by breeding with FLP mouse. This results in cleanly inserted transgene flanked by only the F14 and F15 sites (“TIΔex” allele). Although the structures of TIex allele differ among the site-specific insertion systems employed (e.g. TIex allele 1 and 2; Figure 1), the structure of final TIΔex alleles generated will be identical in all the strategies. Combination of mutant loxPs (JT15/JTZ17 and lox2272) was same as in our original PITT system [6]. Among the multitude of mutant FRT sites reported previously, we chose F14 and F15 (the spacer variants) and FRT-L/R (inverted repeat variant), since F14/F15 pairing was known to be incompatible [9] and thus we anticipated that combination of the spacer variants and the inverted repeat variant would show high recombination efficiency similar to JT15/JTZ17 and lox2272 combinations for the Cre-loxP system [6]. The targeted allele also contains a 43-bp fragment corresponding to human OCT4 gene sequence [described in Hockemeyer et al. [10]] that can serve as a zinc finger nuclease target to aid in insertion of additional sequences for future needs, but the component is not tested yet.Figure 1


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)

Schematic of PITT-mediated targeted insertion using the new seed mice, TOKOM-3. Cre-PITT (via mutant loxP sites) and PhiC31-PITT (via attB/P site) confer insertion of the donor vector containing. DNA Of Interest (DOI), into a target site in the genome, to generate TIex allele 1 or 2 respectively. DOI shown in this example is tdTomato-poly(A) cassette in this figure. Introduction of FLP removes extra sequence in the TIex allele 1 and 4, resulting in generation of TIΔex allele. See Additional file 1: Figures S1, S3 and S4 for other possible outcomes recombination. GTNOP: a cassette containing “eGFP-T2A-Neomycin resistant gene-hOCT4-PolyA”.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Schematic of PITT-mediated targeted insertion using the new seed mice, TOKOM-3. Cre-PITT (via mutant loxP sites) and PhiC31-PITT (via attB/P site) confer insertion of the donor vector containing. DNA Of Interest (DOI), into a target site in the genome, to generate TIex allele 1 or 2 respectively. DOI shown in this example is tdTomato-poly(A) cassette in this figure. Introduction of FLP removes extra sequence in the TIex allele 1 and 4, resulting in generation of TIΔex allele. See Additional file 1: Figures S1, S3 and S4 for other possible outcomes recombination. GTNOP: a cassette containing “eGFP-T2A-Neomycin resistant gene-hOCT4-PolyA”.
Mentions: The overall scheme of i-PITT is illustrated in Figure 1. A new PITT seed mouse (TOKMO-3) under C57BL/6N genetic background was engineered by inserting a landing pad containing loxP derivatives (JT15 and lox2272), attP, and FRT derivatives (F14, F15 and FRT-L) into the Rosa26 locus through ES cell-based targeting. A donor vector carrying tdTomato-pA, loxP derivatives (JTZ17 and lox2272), attB, and FRT derivatives (F14, F15 and FRT-R) was designed to perform site-specific insertion of a transgene (e.g. “tdTomato-pA” in Figure 1). The transgene flanked by the F14 and F15 sites in the donor vector was inserted into target locus of fertilized eggs via i-PITT (Figure 1 and Additional file 1: Figure S1) that offers options for any of the three enzymes Cre, PhiC31 or FLP to choose for targeted insertion. The resulting embryos will exhibit “targeted insertion with extra sequence (TIex)” allele. The strategy is designed in such way that the extra sequences will be flanked by F14 and F15 elements which enable the removal of extra sequences by breeding with FLP mouse. This results in cleanly inserted transgene flanked by only the F14 and F15 sites (“TIΔex” allele). Although the structures of TIex allele differ among the site-specific insertion systems employed (e.g. TIex allele 1 and 2; Figure 1), the structure of final TIΔex alleles generated will be identical in all the strategies. Combination of mutant loxPs (JT15/JTZ17 and lox2272) was same as in our original PITT system [6]. Among the multitude of mutant FRT sites reported previously, we chose F14 and F15 (the spacer variants) and FRT-L/R (inverted repeat variant), since F14/F15 pairing was known to be incompatible [9] and thus we anticipated that combination of the spacer variants and the inverted repeat variant would show high recombination efficiency similar to JT15/JTZ17 and lox2272 combinations for the Cre-loxP system [6]. The targeted allele also contains a 43-bp fragment corresponding to human OCT4 gene sequence [described in Hockemeyer et al. [10]] that can serve as a zinc finger nuclease target to aid in insertion of additional sequences for future needs, but the component is not tested yet.Figure 1

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