Genetically manipulated mice: a powerful tool with unsuspected caveats

Abstract

Although genetic manipulations in mice have provided a powerful tool for investigating gene function in vivo, recent studies have uncovered a number of developmental phenomena that complicate the attribution of phenotype to the specific genetic change. A more realistic approach has been to modulate gene expression and function in a temporal and tissue-specific manner. The most common of these methods, the CreLoxP and tetracycline response systems, are surveyed here and their recently identified shortcomings discussed, along with a less well known system based on the E. coli lac operon and modified for use in mammals. The potential for further complications in interpretation due to hitherto unexpected epigenetic effects involving transfer of RNA or protein in oocytes or sperm is also explored. Given these problems we reiterate the necessity for the use of completely reversible methods that will allow each experimental group of animals to act as their own control. Using these methods with a number of specific modifications to eliminate non-specific effects from random insertion sites and inducer molecules, the full potential of genetic manipulation studies should be realized.

Figure 1. Deletion of intra-LoxP DNA by Cre recombinase

Mice transgenic for the ‘β-galactosidase Floxed stop’ EGFP construct (called FloxStop) express β-galactosidase and not EGFP. When crossed with a Cre mouse the doubly transgenic mice no longer express β-galactosidase due to its deletion (called delLoxP) and the mice are green due to the removal of the stop and de novo EGFP expression.

Figure 2. Reversible regulation of gene activity by the LacO/LacIR system

A, luciferase activity in different tissues from Huntingtin^LacO-luciferase (HDOSluc) mice and repression of activity in HDOSluc mice doubly transgenic for LacI^R. B, de-repression of luciferase activity at 24 and 48 h after addition of 10 mm IPTG in the drinking water.

Figure 3. Epigenetic modification of offspring by transfer of Cre recombinase in oocytes

A, male wild-type Cre-positive mice crossed with FloxStop/FloxStop females produce offspring that are all positive for FloxStop and half are also positive for Cre. Only the Cre-positive mice are green due to the deletion of the stop (delLoxP). B, female wild-type Cre-positive mice crossed with FloxStop/FloxStop males produce offspring that are all positive for Floxtop and half are also positive for Cre. However, all the offspring are green due to the deletion of the stop (delLoxP) even in mice not positive for Cre (circled). The phenotype is due to the transfer of Cre protein from the female into the oocyte where it deletes the Floxed DNA.

Figure 4. Epigenetic modification of offspring by transfer of RNA in sperm

Male Kit^tmAlf/+ mice with white spotted feet and tail tips crossed with wild-type mice produce offspring that all have the white spotted phenotype, even the wild-type mice not carrying the mutant gene (circled). The phenotype is the result of Kit^tmAlf RNA transfer in the sperm from the Kit^tmAlf male to the wild-type mice.