Detecting and Avoiding Problems When Using the Cre-lox System

Abstract

The Cre-lox recombination approach is commonly used to generate cell-specific gene inactivation (or activation). We have noticed that the breeding and genotyping sections of papers utilizing Cre-lox techniques are frequently incomplete. While seemingly straightforward, there are important considerations that need to be implemented in the breeding and genotyping methods to prevent the introduction of experimental confounds. Germline recombination and transient expression of Cre recombinase during development are some examples of the complications that can occur, and conventional genotyping methods may fail to identify these events. In this opinion article, we highlight the importance of testing for unexpected recombination events, suggest strategies to isolate and minimize adverse recombination events, and encourage editors and reviewers to expect more definitive statements regarding the validation of genotyping.

Keywords: Cre recombinase; ectopic gene expression; germline recombination; transgenic mice.

Figure 1

Non-specific expression of a Reporter gene from a genetic cross of Oprk1^Cre/wt ; Rosa26^{lox-stop-lox-tdTomato/wt} with a wild-type mouse. (a) Images of facial area, front limb, hind limb, and front paw (from top to bottom) of a Oprk1^wt/wt Rosa26^{lox-stop-lox-tdTomato/wt} mouse (left) with nonspecific global expression of tdTomato, and its Oprk1^Cre/wt ;Rosa26^{lox-stop-lox-tdTomato/wt} littermate (right). (b) Confocal images of a 40-μm brain section of the dorsal raphe nucleus from the mouse with non-specific expression of tdTomato (left) including in blood vessels (arrow), and its littermate (right) with cell-specific expression of tdTomato (arrows) in kappa opioid receptor expressing neurons taken under the same capture settings.

Figure 2

Detection of unexpected recombination during generation of conditional knockout mice. (a) Top line, is the wild-type (WT) allele with a hypothetical exon (box); below that is the floxed allele with the exon flanked by loxP sites (arrow heads), and below that is the floxed allele after Cre-mediated recombination. The location of PCR primers, a, b, and c is indicated along with the sizes of the PCR products. (b) A genetic cross between a mouse bearing Gene X^Cre/wt and Gene Y^lox/wt and a mouse homozygous for the floxed gene (Gene Y^lox/lox). (c) Hypothetical results from PCR analysis of the offspring that are positive for Cre recombinase using a 2-primer PCR strategy that detects the WT and lox alleles. Lanes 1, 2 show expected results with no unexpected recombination in tail DNA. The WT/lox heteroduplex band may appear after too many PCR cycles. Lanes 3, 5 show the results when there is partial recombination (thin arrow Δ) during early development. Lanes 4, 6 show results when there is complete recombination (thick arrow Δ). Note that with germline or developmental recombination, the lox allele becomes fainter and may even disappear. Recombination can also be observed in Cre-negative offspring (not shown). (d) Hypothetical results using a 3-primer strategy that can detect the Δ allele. Unexpected recombination is revealed by the presence the 200 bp band.

Figure 3

(a) Top line, is the wild-type (WT) allele with a hypothetical exon (box); below that is the floxed allele with the exon flanked by loxP sites (arrow heads), and below that is the floxed allele after Cre-mediated recombination. The location of PCR primers, a, b, and c is indicated along with the sizes of the PCR products. (b) A genetic cross between a mouse bearing Gene X^Cre and Gene Y^Δ/wt and a mouse homozygous for the floxed Gene Y (no Cre). Note that in this breeding scheme, recombination cannot occur in the germline because there is no lox allele in the germline. (c) Hypothetical results from PCR analysis of tail DNA (where recombination is not expected) from Cre-positive offspring. Lanes 1, 2 show expected results with no unexpected recombination in tail DNA. The WT/lox heteroduplex band may appear with too many PCR cycles. Lanes 3, 5 show the results when there is partial recombination (thin arrow Δ) during early development. Lanes 4, 6 show results when there is complete recombination (thick arrow Δ) during early development. The results are the same as when the parent with Gene X^Cre carries Gene Y^lox/wt allele (Figure 2D); however, in that case one does not know whether recombination occurred in germline or during early development. Recombination can also be observed in Cre-negative offspring (not shown). (d) PCR results from a genetic cross of the type shown in B; Note the presence of the Δ allele in lanes 2, 3, and 4. In lane 2, there must have been complete recombination of the lox allele during early development because WT and Δ alleles are not possible from this cross; lanes 3 and 4 reveal partial recombination of the lox allele.