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

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.

Figure 1

LE/RE mutant lox system. (a) Wild-type loxP sequence. The loxP site is composed of an asymmetric 8-bp spacer flanked by 13-bp inverted repeats that the Cre recombinase recognizes and binds to. (b) Integrative recombination with LE/RE mutant lox sites. The asterisks represent the mutations. RE mutants have mutations in the right inverted repeat region, and LE mutants have mutations in the left inverted repeat region. Through recombination between LE and RE mutant lox sites, a wild-type loxP site and a double mutant lox site carrying mutations in both ends are produced. The double mutant lox site has a lower affinity for the Cre enzyme; therefore, recombination of the product rarely occurs, and the recombination reaction tends toward integration. (c) Sequences of mutant lox sites used in this study. Lox71 carries 5 bp of mutation in the left element. The sequence of the right element of lox66 is complementary to the left element sequence of lox71. Mutated sequences are indicated by bold and are underlined.

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.

Figure 3

Relative integration efficiency. The efficiency obtained with the lox66 integration plasmid arbitrarily set at 1.

Figure 4

Strategy to evaluate the stability of double mutant lox. In the Ttr-KO41 line, the lox71-Pgk-neo-loxP-pA-lox2272 cassette was inserted in the first exon of the mouse Ttr gene. Replacement vectors carried the RE mutant lox-LacZ-pA-Pgk-Pac-loxP. The Ttr-KO41 line was coelectroporated with the each replacement vector and the Cre-expressing plasmid; it was then selected with puromycin. Colonies were picked, and six sub-clones (71/66-P, 71/JTZ-P, 71/KR1-P, 71/KR2-P, 71/KR3-P, and 71/KR4-P) carrying different double mutant lox were established. To induce stable Cre-expression, the CAG-Cre-IRES-Hyg transgene was introduced into each sub-clone and selected with hygromycin. For the short-term stability assay, electroporated cells were divided into two plates, and after 5 days of hyg selection, one plate was selected with puro and the other with normal medium. The ratio of hyg^R puro^S colonies to hyg^R colonies represents the short-term stability of the double mutant lox site. For the long-term stability assay, Cre-expressing sub-lines with high levels were selected and passaged 9~11 times. Genomic DNA was prepared after 4, 7, 9, and 11 passages, and recombination at each stage was detected with Southern blotting.

Figure 5

Short-term stability of double mutant lox sites. The ratios of hyg^R puro^S colonies to hyg^R colonies from four independent electroporations are represented. Bars indicate the means.

Figure 6

Northern blot analysis of Cre-expressing sub-lines. (a) Six sub-lines carrying the CAG-Cre-IRES-Hyg cassette were isolated from each of three double-mutant lox lines: 71/66-P, 71/JTZ-P, and 71/KR3-P. Ten micrograms of total RNA were subjected to electrophoresis and hybridization with a Cre probe. The two highest Cre-expressing lines selected from each of the six sub-lines are indicated by a circle. (b) Selected sub-lines were passaged 11 times, and RNAs from 1st, 7th, and 11th passage were prepared. Four micrograms of total RNAs were subjected to electrophoresis and hybridization with a Cre probe. The expression levels of Cre were maintained during passages.

Figure 7

Excising recombination between double mutant lox and wild-type loxP in strongly Cre-expressing sub-lines. (a) Restriction endonuclease map of the targeted Ttr locus before (Ttr LacZ-KI allele) and after (Excised allele) Cre-mediated recombination. The pA signal of the mouse Pgk gene was used as a probe (indicated by the solid line). Fragment sizes are indicated. Bg, BglII; Sc, ScaI. (b) Southern blot analysis of Cre-expressing sub-lines. P represents the parental line before introducing the CAG-Cre-IRES-Hyg cassette. The numbers indicate passage numbers from the original cell stock. The positions of the bands from the nonexcised LacZ allele (LacZ), excised allele (Excised), and endogenous Pgk gene (Endo) are indicated by arrowheads. (c) Relative band intensities of the LacZ allele (hatched line) and excised allele (solid line) to the band intensities of the endogenous Pgk gene. The rate of excised band [ratio of excised band intensity to total (LacZ + excised) intensity] was calculated and is indicated by the gray line.

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.