Brian Sauer

Cre‐mediated site‐specific recombination in zebrafish embryos

Cre‐mediated site‐specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) –Cre fusion protein. The EGFP–Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post–heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70‐EGFP–cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP–Cre expression. Hence, our data suggests that Cre‐mediated recombination is a viable means of manipulating gene expression in zebrafish. Developmental Dynamics 233:1366–1377, 2005.

Cre/Iox: One more step in the taming of the genome

Cre recombinase has become an important tool in the precise manipulation of the genome, and its adoption has led to the development of increasingly accurate mouse models for the understanding of gene function. Although much of current work exploits the alacrity and precision with which Cre catalyzes excisive DNA recombination, Cre also is adept at the insertion of heterologous DNA into the genome. The precision and efficiency with which Cre can target DNA to a predesignated locus in the genome promises to facilitate understanding of mutant genes and allelic variants in their natural chromosomal context.

Cre Recombination‐Mediated Cassette Exchange for Building Versatile Transgenic Human Embryonic Stem Cells Lines

To circumvent the silencing effect of transgene expression in human embryonic stem cells (hESCs), we employed the Cre recombination‐mediated cassette exchange strategy to target the silencing‐resistant site in the genome. We have identified new loci that sustain transgene expression during stem cell expansion and differentiation to cells representing the three germ layers in vitro and in vivo. The built‐in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites, using the cell‐permeable Cre protein transduction method, resulting in successful generation of new hESC lines with constitutive functional gene expression, inducible transgene expression, and lineage‐specific reporter gene expression. This strategy and the master cell lines allow for rapid production of transgenic hESC lines in ordinary laboratories. Stem Cells 2009;27:1032–1041

Non-contact positions impose site selectivity on Cre recombinase

A first step in Cre-mediated site-specific DNA recombination is binding to the two 13 bp repeats of the 34 bp site loxP. Several nucleotides within loxP do not directly contact the bound enzyme, yet mutation at two of these base pairs, at positions 11 and 12 in each repeat, results in a 100 000-fold reduction in recombination. To understand better how Cre selects DNA sequences for recombination, we combined DNA shuffling mutagenesis and a forward selection strategy to obtain Cre mutants that recombine at 100% efficiency a mutant loxK2 site carrying these dinucleotide changes. The role of the several mutations found in these Cre isolates was analyzed both in vivo and biochemically with purified enzymes. A single mutation at E262 accounts for most but not all of the enhanced activity at loxK2. Secondary mutations act in one or more of three ways: enhancement of loxK2 binding, accelerated synthesis of Cre in vivo or faster DNA recombination at the alternative spacer region present in loxK2. Systematic analysis of all 20 natural amino acids at position E262 shows that the naturally occurring glutamate residue at this position provides the optimal balance of efficiency of recombination at loxP and maximal discrimination against loxK2.

Analysis of Cre-loxP interaction by surface plasmon resonance: influence of spermidine on cooperativity.

To study target site selectivity of one important class of DNA-binding proteins, site-specific DNA recombinases, we developed an automated real-time kinetic assay based on surface plasmon resonance (BIACORE) and formulated a curve-fitting model that takes into account cooperative interactions. Monitoring the interaction between the Cre DNA recombinase and its specific target site loxP by BIACORE, we found that Cre associates with loxP tightly and highly cooperatively. We observed that the cooperative moment of the Cre-loxP interaction is strongly dependent on the concentration of spermidine, a small polyamine influencing DNA conformation. Thus, DNA conformation can have a profound impact on substrate recognition and subsequent recombination.

Transgenic mice over-expressing endothelin-1 in testis transactivated by a Cre/loxP system showed decreased testicular capillary blood flow

It is generally believed that too high or low levels of endothelin-1 (ET-1), a strong vasoconstrictor, may be detrimental to animals. Therefore, in order to understand the in vivo function of ET-1, we used a conditional transgenic approach, Cre/loxP recombination system, to generate transgenic mice that over-express ET-1 in a tissue-specific manner. In such a strategy a single transgenic mouse line, ELSE, was initially generated where a general promoter, human elongation factor 1α (hEF1α) promoter, was used to drive the expression of a loxP-flanked sequence containing the lacZ reporter gene and a STOP cassette before the ET-1 cDNA, the recombinational competency of which was confirmed in an Escherichia coli test system. In ELSE mice, expression of the reporter lacZ was limited to spermatozoa and spermatogonia as well as Sertoli, Leydig and endothelial cells in the testis, thus confirming the suitability of these mice for the generation of testes-limited ET-1 expression. To generate transgenic progeny with ET-1 over-expression in the testis (successful recombination, ELSE/ELT), ELSE mice were mated with EIIa-cre mice expressing Cre recombinase in pre-implantation mouse embryos. These ELSE/ELT mice exhibiting testis-specific ET-1 over-expression had normal reproductive function and showed no obvious alterations in gross testicular morphology. Although over-expression of ET-1 leads to reduction of testicular blood flow, young adult ELSE/ELT mice showed no obvious signs of inflammation, fibrosis or abnormal proliferation of cells in the testes of young ELSE/ELT mice by histochemical analyses.