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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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PITT using TOKOM-3 seed mice. (A) Schematic diagram of the PITT injection experiment. Donor vector containing DOI is injected into the fertilized eggs harboring landing pad together with iCre and/or PhiC31o mRNA. (B) Successful targeted transgenesis in blastocysts derived from zygotes injected with pBER + iCre mRNA + PhiC31o mRNA. (C) Successful targeted transgenesis in day 13.5 fetuses. Zygotes/fetuses exhibiting red fluorescence indicate successful targeted insertion of DOI. (D, E) The PITT results in blastocysts (D) and fetuses/pups (E).
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Fig3: PITT using TOKOM-3 seed mice. (A) Schematic diagram of the PITT injection experiment. Donor vector containing DOI is injected into the fertilized eggs harboring landing pad together with iCre and/or PhiC31o mRNA. (B) Successful targeted transgenesis in blastocysts derived from zygotes injected with pBER + iCre mRNA + PhiC31o mRNA. (C) Successful targeted transgenesis in day 13.5 fetuses. Zygotes/fetuses exhibiting red fluorescence indicate successful targeted insertion of DOI. (D, E) The PITT results in blastocysts (D) and fetuses/pups (E).

Mentions: The PITT efficiency for Cre- or PhiC31-based systems was assessed by injecting zygotes, culturing them until blastocysts, and subsequent analysis for targeted insertion (Figure 3A and B). Targeted insertion was detected in 28.6% or 16.7% of developed blastocysts (9.0% or 7.6% of total injected zygotes) when iCre mRNA or PhiC31o mRNA were injected (Figure 3D). Interestingly, co-injection of iCre and PhiC31o mRNAs resulted in increased insertion efficiency of up to 36.8% of developed blastocysts (up to 15.1% of total injected zygotes) (Figure 3D). Notably, insertion rate as high as 61.5% among developed blastocysts was achieved in a single experiment (Experiment 7 in Additional file 1: Table S2). Based on these results, we decided to follow a co-injection strategy using iCre mRNA and PhiC31o mRNA at concentrations 0.5 ng/μl and 7.5 ng/μl respectively in subsequent experiments.Figure 3


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)

PITT using TOKOM-3 seed mice. (A) Schematic diagram of the PITT injection experiment. Donor vector containing DOI is injected into the fertilized eggs harboring landing pad together with iCre and/or PhiC31o mRNA. (B) Successful targeted transgenesis in blastocysts derived from zygotes injected with pBER + iCre mRNA + PhiC31o mRNA. (C) Successful targeted transgenesis in day 13.5 fetuses. Zygotes/fetuses exhibiting red fluorescence indicate successful targeted insertion of DOI. (D, E) The PITT results in blastocysts (D) and fetuses/pups (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: PITT using TOKOM-3 seed mice. (A) Schematic diagram of the PITT injection experiment. Donor vector containing DOI is injected into the fertilized eggs harboring landing pad together with iCre and/or PhiC31o mRNA. (B) Successful targeted transgenesis in blastocysts derived from zygotes injected with pBER + iCre mRNA + PhiC31o mRNA. (C) Successful targeted transgenesis in day 13.5 fetuses. Zygotes/fetuses exhibiting red fluorescence indicate successful targeted insertion of DOI. (D, E) The PITT results in blastocysts (D) and fetuses/pups (E).
Mentions: The PITT efficiency for Cre- or PhiC31-based systems was assessed by injecting zygotes, culturing them until blastocysts, and subsequent analysis for targeted insertion (Figure 3A and B). Targeted insertion was detected in 28.6% or 16.7% of developed blastocysts (9.0% or 7.6% of total injected zygotes) when iCre mRNA or PhiC31o mRNA were injected (Figure 3D). Interestingly, co-injection of iCre and PhiC31o mRNAs resulted in increased insertion efficiency of up to 36.8% of developed blastocysts (up to 15.1% of total injected zygotes) (Figure 3D). Notably, insertion rate as high as 61.5% among developed blastocysts was achieved in a single experiment (Experiment 7 in Additional file 1: Table S2). Based on these results, we decided to follow a co-injection strategy using iCre mRNA and PhiC31o mRNA at concentrations 0.5 ng/μl and 7.5 ng/μl respectively in subsequent experiments.Figure 3

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