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Brian Sauer

Publications and source records attributed to Brian Sauer.

12 recordsLinked to original sources
PubMed2005

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.

Animals↗
PubMed2004

DNA recombination with a heterospecific Cre homolog identified from comparison of the pac-c1 regions of P1-related phages.

Sequencing of the 7 kb immC region from four P1-related phages identified a novel DNA recombinase that exhibits many Cre-like characteristics, including recombination in mammalian cells, but which has a distinctly different DNA specificity. DNA sequence comparison to the P1 immC region showed that all phages had related DNA terminase, C1 repressor and DNA recombinase genes. Although these genes from phages P7, phi(w39) and p15B were highly similar to those from P1, those of phage D6 showed significant divergence. Moreover, the D6 sequence showed evidence of DNA deletion and substitution in this region relative to the other phages. Characterization of the D6 site-specific DNA recombinase (Dre) showed that it was a tyrosine recombinase closely related to the P1 Cre recombinase, but that it had a distinct DNA specificity for a 32 bp DNA site (rox). Cre and Dre are heterospecific: Cre did not catalyze recombination at rox sites and Dre did not catalyze recombination at lox sites. Like Cre, Dre catalyzed both integrative and excisive recombination and required no other phage-encoded proteins for recombination. Dre-mediated recombination in mammalian cells showed that, like Cre, no host bacterial proteins are required for efficient Dre-mediated site-specific DNA recombination.

Animals↗
PubMed2004

Functional mapping of Cre recombinase by pentapeptide insertional mutagenesis.

Cre is a site-specific recombinase from bacteriophage P1. It is a member of the tyrosine integrase family and catalyzes reciprocal recombination between specific 34-bp sites called loxP. To analyze the structure-function relationships of this enzyme, we performed large scale pentapeptide insertional mutagenesis to generate insertions of five amino acids at random positions in the protein. The high density of insertion mutations into Cre allowed us to identify an unexpected degree of functional tolerance to insertions into the 4-5 beta-hairpin and into the loop between helices J and K (both of which contact the DNA in the minor groove) and also into helix A. The phenotypes of the majority of inserts allowed us to confirm a variety of predictions made on the basis of sequence conservation, known three-dimensional structure, and proposed catalytic mechanism. In particular, most insertions into conserved regions or secondary structure elements inactivated Cre, and most insertions located in nonconserved, unstructured regions preserved Cre activity. Less expectedly, the non-conserved and poorly structured loops and linkers between helices A-B, E-F, and M-N did not tolerate insertions, thus identifying these as critical regions for recombinase activity. We purified and characterized in vitro several representatives of these "unexpected" Cre insertion mutants. The role of those regions in the recombination process is discussed.

Amino Acid Sequence↗
PubMed2004

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 1alpha (hEF1alpha) 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.

Animals↗
PubMed2004

P2 growth restriction on an rpoC mutant is suppressed by alleles of the Rz1 homolog lysC.

Escherichia coli strain 397c carries a temperature-sensitive mutation, rpoC397, that removes the last 50 amino acids of the RNA polymerase beta' subunit and is nonpermissive for plating of bacteriophage P2. P2 gor mutants productively infect 397c and define a new gene, lysC, encoded by a reading frame that extensively overlaps the P2 lysis accessory gene, lysB. The unusual location of lysC with respect to lysB is reminiscent of the Rz/Rz1 lysis gene pair of phage lambda. Indeed, coexpression of lysB and lysC complemented the growth defect of lambda Rz/Rz1 null mutants, indicating that the LysB/C pair is similar to Rz/Rz1 in both gene arrangement and function. Cells carrying the rpoC397 mutation exhibited an early onset of P2-induced lysis, which was suppressed by the gor mutation in lysC. We propose that changes in host gene expression resulting from the rpoC397 mutation result in changes in the composition of the bacterial cell wall, making the cell more susceptible to P2-mediated lysis and preventing accumulation of progeny phage sufficient for plaque formation.

Amino Acid Sequence↗
PubMed2003

Double lox targeting for neural cell transgenesis.

ES cells differentiated along the neural lineage in vitro are an attractive model system. Here we have developed ES cell lines that are suitable for inserting transgenes at a single chromosomal site. ES cell line CE1 (for Cassette Exchange) contains one "acceptor" module (CE1) that allows for efficient double lox targeting. The site is also permissive for gene expression in neural progenitor cells, as well as differentiated neurons and glia. Line CE2 was derived by swapping a puromycin resistance cassette into CE1. Neural progenitors derived from this line are puromycin-resistant. A beta-actin/GFP expression cassette was inserted into the CE1 site to create CE3. The CE3 cell line was differentiated into neural cells and displayed strong EGFP expression in neural progenitors, differentiated neurons and glia. Differentiated CE3 ES cells (4-/4+ RA) were transplanted into the injured rat somatosensory cortex where many of the transplanted cells survived and differentiated into neurons expressing GFP. This strategy for creating sets of transgenic lines with multiple cassettes inserted into a single chromosomal site provides a powerful tool for studying development and function of ES cell-derived neural cells. Many of these will be useful in transplantation research.

Animals↗
PubMed2003

Far-upstream elements are dispensable for tissue-specific proenkephalin expression using a Cre-mediated knock-in strategy.

Several cis-regulatory DNA elements are present in the 5' upstream regulatory region of the enkephalin gene (ENK) promoter. To determine their role in conferring organ-specificity of ENK expression in mice and to circumvent the position effects from random gene insertion that are known to often frustrate such analysis in transgenic mice, we used a Cre-mediated gene knock-in strategy to target reporter constructs to a "safe haven" loxP-tagged locus in the hypoxanthine phosphoribosyltransferase (HPRT) gene. Here we report reliable and reproducible reporter gene expression under the control of the 5' upstream regulatory region of the mouse ENK gene in gene-modified mice using this Cre-mediated knock-in strategy. Comparison of two 5'ENK regulatory regions (one with and the other without known cis-regulatory DNA elements) in the resulting adult mice showed that conserved far-upstream cis-regulatory DNA elements are dispensable for correct organ-specific gene expression. Thus the proximal 1.4 kb of the murine ENK promoter region is sufficient for organ-specificity of ENK gene expression when targeted to a safe-haven genomic locus. These results suggest that conservation of the far-upstream DNA elements serves more subtle roles, such as the developmental or cell-specific expression of the ENK gene.

5' Flanking Region↗
PubMed2002

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.

Base Sequence↗
PubMed2002

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.

Amino Acid Sequence↗
PubMed2002

Cre/lox: 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.

Animals↗