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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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Evaluation of recombination efficiency. (a) Schematic experimental strategy. Four ES cell lines carrying a single copy of CAG promoter-lox71-bsr-pA were established and coelectroporated with an integration plasmid and the Cre expression vector, pCAGGS-Cre. After G418 selection, the colonies were stained with X-gal, and the percentage of blue colonies was scored as the site-specific integration efficiency. (b) Example of X-gal staining. Bs17 ES cells were coelectroporated with pCAGGS-Cre and pKR3NZneo, selected with G418 for 1 week, and then stained with X-gal. (c) Magnified photo of blue colonies. Most of blue colonies were stained uniformly. (d) X-gal stained plate electroporated without pCAGGS-Cre. Bs17 ES cells were electroporated with only pKR3NZneo. No blue colony appeared.
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Figure 2: Evaluation of recombination efficiency. (a) Schematic experimental strategy. Four ES cell lines carrying a single copy of CAG promoter-lox71-bsr-pA were established and coelectroporated with an integration plasmid and the Cre expression vector, pCAGGS-Cre. After G418 selection, the colonies were stained with X-gal, and the percentage of blue colonies was scored as the site-specific integration efficiency. (b) Example of X-gal staining. Bs17 ES cells were coelectroporated with pCAGGS-Cre and pKR3NZneo, selected with G418 for 1 week, and then stained with X-gal. (c) Magnified photo of blue colonies. Most of blue colonies were stained uniformly. (d) X-gal stained plate electroporated without pCAGGS-Cre. Bs17 ES cells were electroporated with only pKR3NZneo. No blue colony appeared.

Mentions: To test whether these RE lox sites could promote recombination efficiency in ES cells, we used the same strategy as described previously [14](Figure 2a). For the chromosomal target lox71 site, the CAG promoter-lox71-bsr-pA was introduced into ES cells. We isolated four ES clones—Bs1, Bs17, Bs19, and Bs21—that carried a single copy of the chromosomal target construct. Integration vectors comprised an RE lox site on the 5' side of the promoterless NLSlacZ gene and a selection marker gene, MC1-neo-pA. The ES clones were coelectroporated with the integration vector and the Cre-expression vector pCAGGS-Cre (in their circular forms) and then selected with G418. The cre gene was transiently expressed, mediating site-specific recombination between the chromosomal lox71 and the RE lox on the integration plasmid; this resulted in site-specific integration. The neo cassette is active regardless of the integration site; therefore, both random integrants and site-specific recombinants become drug resistant. However, in the present study, only the colonies where site-specific integration had occurred were stained blue with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) because the NLSLacZ gene was inserted downstream of the CAG promoter through Cre-mediated recombination. As shown in Figure 2b and 2c, most of the blue colonies were uniformly blue, indicating that recombination occurred in the early stage of colony formation. The percentage of blue colonies represented the frequency of site-specific integration. When only the integration vector was electroporated, no blue colonies appeared (Figure 2d).


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)

Evaluation of recombination efficiency. (a) Schematic experimental strategy. Four ES cell lines carrying a single copy of CAG promoter-lox71-bsr-pA were established and coelectroporated with an integration plasmid and the Cre expression vector, pCAGGS-Cre. After G418 selection, the colonies were stained with X-gal, and the percentage of blue colonies was scored as the site-specific integration efficiency. (b) Example of X-gal staining. Bs17 ES cells were coelectroporated with pCAGGS-Cre and pKR3NZneo, selected with G418 for 1 week, and then stained with X-gal. (c) Magnified photo of blue colonies. Most of blue colonies were stained uniformly. (d) X-gal stained plate electroporated without pCAGGS-Cre. Bs17 ES cells were electroporated with only pKR3NZneo. No blue colony appeared.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Evaluation of recombination efficiency. (a) Schematic experimental strategy. Four ES cell lines carrying a single copy of CAG promoter-lox71-bsr-pA were established and coelectroporated with an integration plasmid and the Cre expression vector, pCAGGS-Cre. After G418 selection, the colonies were stained with X-gal, and the percentage of blue colonies was scored as the site-specific integration efficiency. (b) Example of X-gal staining. Bs17 ES cells were coelectroporated with pCAGGS-Cre and pKR3NZneo, selected with G418 for 1 week, and then stained with X-gal. (c) Magnified photo of blue colonies. Most of blue colonies were stained uniformly. (d) X-gal stained plate electroporated without pCAGGS-Cre. Bs17 ES cells were electroporated with only pKR3NZneo. No blue colony appeared.
Mentions: To test whether these RE lox sites could promote recombination efficiency in ES cells, we used the same strategy as described previously [14](Figure 2a). For the chromosomal target lox71 site, the CAG promoter-lox71-bsr-pA was introduced into ES cells. We isolated four ES clones—Bs1, Bs17, Bs19, and Bs21—that carried a single copy of the chromosomal target construct. Integration vectors comprised an RE lox site on the 5' side of the promoterless NLSlacZ gene and a selection marker gene, MC1-neo-pA. The ES clones were coelectroporated with the integration vector and the Cre-expression vector pCAGGS-Cre (in their circular forms) and then selected with G418. The cre gene was transiently expressed, mediating site-specific recombination between the chromosomal lox71 and the RE lox on the integration plasmid; this resulted in site-specific integration. The neo cassette is active regardless of the integration site; therefore, both random integrants and site-specific recombinants become drug resistant. However, in the present study, only the colonies where site-specific integration had occurred were stained blue with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal) because the NLSLacZ gene was inserted downstream of the CAG promoter through Cre-mediated recombination. As shown in Figure 2b and 2c, most of the blue colonies were uniformly blue, indicating that recombination occurred in the early stage of colony formation. The percentage of blue colonies represented the frequency of site-specific integration. When only the integration vector was electroporated, no blue colonies appeared (Figure 2d).

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