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Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells.

Araki K, Okada Y, Araki M, Yamamura K - BMC Biotechnol. (2010)

Bottom Line: We previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells.Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71.The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Resource Development and Analysis, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan. arakimi@gpo.kumamoto-u.ac.jp

ABSTRACT

Background: Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of lox sites with mutations in the LE or RE of the lox sequence has previously been developed. Recombination between LE and RE mutant lox produces a wild-type loxP site as well as an LE+RE double mutant lox site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells. Recently, other LE/RE mutant lox sites showing higher recombination efficiency in Escherichia coli have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.

Results: Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71. All of the RE mutant lox sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant lox site, under continuous and strong Cre activity in ES cells. Two RE mutants, loxJTZ17 and loxKR3, produced more stable LE+RE double mutant lox than did the lox66/71 double mutant.

Conclusion: The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.

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Related in: MedlinePlus

Relation of excision and Cre expression level. The northern blot X-ray film of Figure 6b was subjected to densitometry; the average of band intensities of samples from the 1st, 7th, and 11th passages were calculated; and the relative band intensity to 71/JTX-P-Cre 4, which showed the lowest expression, was calculated and plotted on the x-axis. The highest value of the excised allele rate calculated in Figure 7c was plotted on the y-axis. In 71/66-P-Cre clones, the recombination rate was apparently higher than in other clones.
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Figure 8: Relation of excision and Cre expression level. The northern blot X-ray film of Figure 6b was subjected to densitometry; the average of band intensities of samples from the 1st, 7th, and 11th passages were calculated; and the relative band intensity to 71/JTX-P-Cre 4, which showed the lowest expression, was calculated and plotted on the x-axis. The highest value of the excised allele rate calculated in Figure 7c was plotted on the y-axis. In 71/66-P-Cre clones, the recombination rate was apparently higher than in other clones.

Mentions: In order to compare Cre expression levels in these Cre sub-clones, the band intensities shown in Figure 6b were measured by densitometry, and Cre expression level relative to 71/JTZ-P-Cre 4 was calculated. Figure 8 shows a scatter plot of the rate of excision (y-axis) and Cre expression level (x-axis) in each sub-clone. In 71/JTZ-P-Cre and 71/KR3-P-Cre clones, excision rate and Cre expression levels seemed to have a linear relationship with a similar correlation coefficient, suggesting that lox71/JTZ17 and lox71/KR3 have an in-affinity (stability) level similar to Cre protein. On the other hand, 71/66-P-Cre clones showed 2-4 times higher excision levels than did 71/JTZ-P-Cre and 71/KR3-P-Cre clones.


Comparative analysis of right element mutant lox sites on recombination efficiency in embryonic stem cells.

Araki K, Okada Y, Araki M, Yamamura K - BMC Biotechnol. (2010)

Relation of excision and Cre expression level. The northern blot X-ray film of Figure 6b was subjected to densitometry; the average of band intensities of samples from the 1st, 7th, and 11th passages were calculated; and the relative band intensity to 71/JTX-P-Cre 4, which showed the lowest expression, was calculated and plotted on the x-axis. The highest value of the excised allele rate calculated in Figure 7c was plotted on the y-axis. In 71/66-P-Cre clones, the recombination rate was apparently higher than in other clones.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2865440&req=5

Figure 8: Relation of excision and Cre expression level. The northern blot X-ray film of Figure 6b was subjected to densitometry; the average of band intensities of samples from the 1st, 7th, and 11th passages were calculated; and the relative band intensity to 71/JTX-P-Cre 4, which showed the lowest expression, was calculated and plotted on the x-axis. The highest value of the excised allele rate calculated in Figure 7c was plotted on the y-axis. In 71/66-P-Cre clones, the recombination rate was apparently higher than in other clones.
Mentions: In order to compare Cre expression levels in these Cre sub-clones, the band intensities shown in Figure 6b were measured by densitometry, and Cre expression level relative to 71/JTZ-P-Cre 4 was calculated. Figure 8 shows a scatter plot of the rate of excision (y-axis) and Cre expression level (x-axis) in each sub-clone. In 71/JTZ-P-Cre and 71/KR3-P-Cre clones, excision rate and Cre expression levels seemed to have a linear relationship with a similar correlation coefficient, suggesting that lox71/JTZ17 and lox71/KR3 have an in-affinity (stability) level similar to Cre protein. On the other hand, 71/66-P-Cre clones showed 2-4 times higher excision levels than did 71/JTZ-P-Cre and 71/KR3-P-Cre clones.

Bottom Line: We previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells.Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71.The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Resource Development and Analysis, Kumamoto University, Honjo 2-2-1, Kumamoto 860-0811, Japan. arakimi@gpo.kumamoto-u.ac.jp

ABSTRACT

Background: Cre-mediated site-specific integrative recombination in mouse embryonic stem (ES) cells is a useful tool for genome engineering, allowing precise and repeated site-specific integration. To promote the integrative reaction, a left element/right element (LE/RE) mutant strategy using a pair of lox sites with mutations in the LE or RE of the lox sequence has previously been developed. Recombination between LE and RE mutant lox produces a wild-type loxP site as well as an LE+RE double mutant lox site, which has mutations in both sides and less affinity to Cre, resulting in stable integration. We previously demonstrated successful integrative recombination using lox71 (an LE mutant) and lox66 (an RE mutant) in ES cells. Recently, other LE/RE mutant lox sites showing higher recombination efficiency in Escherichia coli have been reported. However, their recombination efficiency in mammalian cells remains to be analyzed.

Results: Using ES cells, we compared six RE mutant lox sites, focusing on their recombination efficiency with lox71. All of the RE mutant lox sites showed similar recombination efficiency. We then analyzed the stability of the recombined product, i.e., the LE+RE double mutant lox site, under continuous and strong Cre activity in ES cells. Two RE mutants, loxJTZ17 and loxKR3, produced more stable LE+RE double mutant lox than did the lox66/71 double mutant.

Conclusion: The two mutant RE lox sites, loxJTZ17 and loxKR3, are more suitable than lox66 for Cre-mediated integration or inversion in ES cells.

Show MeSH
Related in: MedlinePlus