The somatomedin hypothesis proposed that insulin-like growth factor I (IGF-I) was a hepatically derived circulating mediator of growth hormone and is a crucial factor for postnatal growth and development. To reassess this hypothesis, we have used the Cre͞loxP recombination system to delete the igf1 gene exclusively in the liver. igf1 gene deletion in the liver abrogated expression of igf1 mRNA and caused a dramatic reduction in circulating IGF-I levels. However, growth as determined by body weight, body length, and femoral length did not differ from wild-type littermates. Although our model proves that hepatic IGF-I is indeed the major contributor to circulating IGF-I levels in mice it challenges the concept that circulating IGF-I is crucial for normal postnatal growth. Rather, our model provides direct evidence for the importance of the autocrine͞paracrine role of IGF-I.

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The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra-and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(P04)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis ofDNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.

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An efficient and accurate method for controlled in vivo transgene modulation by site-directed recombination is described. Seven transgenic mouse founder lines were produced carrying the murine lens-specific aA-crystallin promoter and the simian virus 40 large tumor-antigen gene sequence, separated by a 1.3-kilobase-pair Stop sequence that contains elements preventing expression of the large tumorantigen gene and Cre recombinase recognition sites. Progeny from two of these lines were mated with transgenic mice expressing the Cre recombinase under control of either the murine aA-crystallin promoter or the human cytomegalovirus promoter. AU double-transgenic offspring developed lens tumors. Subsequent analysis confirmed that tumor formation resulted from large tumor-antigen activation via site-specific, Cre-mediated deletion of Stop sequences.A desired goal of transgene technology is efficient and accurate manipulation of DNA sequences after their integration in the germ line. DNA recombinases that mediate integration or excision of sequences at specific recognition sites in both prokaryotic (1-5) and eukaryotic (6-10) systems are well suited for this purpose. The bacteriophage P1 recombinase Cre catalyzes reciprocal recombination at a specific locus ofcrossing over (lox) (11-16). The lox sequence is composed of two 13-base-pair (bp) inverted repeats separated by an 8-bp spacer region. Upon binding to the inverted repeats, Cre synapses with a second lox site and then cleaves the DNA in the spacer region to initiate strand exchange with the synapsed lox partner. No additional factors are required in the recombination.In this study, we examine the potential of the cre/lox system to activate a dormant transgene in the mouse. The simian virus 40 (SV40) large tumor antigens (TAgs) directed to the lens by a murine aA-crystallin promoter (maA) cause malignant lens tumors (17). We inserted between maA and TAg a specially designed Stop sequence that prevents gene expression and is flanked by lox sequences. By crossing the dormant TAg transgenic mouse lines with Cre-expressing transgenic lines, we report here that the Cre protein recognizes the lox sites of the maA-Stop-TAg transgene and recombines the two lox sequences, thereby removing Stop and activating TAg. Our studies show that targeted transgene modification in the mouse can be performed efficiently and accurately with a prokaryotic recombinase.

We have used gene targeting to create a mouse model of glycogen storage disease type II, a disease in which distinct clinical phenotypes present at different ages. As in the severe human infantile disease (Pompe Syndrome), mice homozygous for disruption of the acid ␣-glucosidase gene (6 neo /6 neo ) lack enzyme activity and begin to accumulate glycogen in cardiac and skeletal muscle lysosomes by 3 weeks of age, with a progressive increase thereafter. By 3.5 weeks of age, these mice have markedly reduced mobility and strength. They grow normally, however, reach adulthood, remain fertile, and, as in the human adult disease, older mice accumulate glycogen in the diaphragm. By 8 -9 months of age animals develop obvious muscle wasting and a weak, waddling gait. This model, therefore, recapitulates critical features of both the infantile and the adult forms of the disease at a pace suitable for the evaluation of enzyme or gene replacement. In contrast, in a second model, mutant mice with deletion of exon 6 (⌬6/⌬6), like the recently published acid ␣-glucosidase knockout with disruption of exon 13

The procaryotic cre-lox site-specific recombination system of coliphage P1 was shown to function in an efficient manner in a eucaryote, the yeast Saccharomyces cerevisiae. The cre gene, which codes for a site-specific recombinase, was placed under control of the yeast GAL] promoter. lox sites flanking the LEU2 gene were integrated into two different chromosomes in both orientations. Excisive recombination at the lox sites (as measured by loss of the LEU2 gene) was promoted efficiently and accurately by the Cre protein and was dependent upon induction by galactose. These results demonstrate that a procaryotic recombinase can enter a eucaryotic nucleus and, moreover, that the ability of the Cre recombinase to perform precise recombination events on the chromosomes of S. cerevisiae is unimpaired by chromatin strujcture.In general, the genome of eucaryotic cells is larger and more structurally complex than that of procaryotic cells. Moreover, the eucaryotic genome is composed of multiple linear chromosomes, whereas bacteria tend to have a single circular chromosome. A distinctive eucaryotic feature is that the genomic DNA is organized into nucleosomes by intimate association with histones and other proteins to form chromatin. Treatment of chromatin with micrococcal nuclease results in a characteristic nucleosome repeat structure (16). Moreover, chromatin can exist in both transcriptionally active and inactive forms. This difference is most likely due to differences in accessibility of the DNA, as measured by susceptibility to nuclease digestion (34).The ability of an enzyme to access DNA in chromatin may not be a property intrinsic to its eucaryotic source. Certainly eucaryotic DNA polymerases, RNA polymerases, and topoisomerases can act on naked DNA in vitro. Conversely, procaryotic proteins such as the lexA repressor and the EcoRI endonuclease can act in vivo at specific DNA sequences on the chromosomes of the yeast Saccharomyces cerevisiae (3, 5). These observations suggest that procaryotic proteins may also be able to conduct more sophisticated transactions on the DNA of a eucaryotic chromosome. For instance, can DNA recombination events in eucaryotes be promoted by a bacterial protein? Such events demand not only recognition of DNA sequences but also synapsis, DNA cleavage, strand exchange, and religation. In particular, can a site-specific recombination system of Escherichia coli function in a eucaryotic cell? The cre-lox site-specific recombination system of coliphage P1 is well suited to answer such questions.Phage P1 encodes an efficient site-specific recombination system consisting of a short asymmetric DNA sequence called loxP and a 38-kilodalton protein called Cre (1,12,29). The loxP site is a 34-base-pair sequence composed of two 13-base-pair inverted repeats separated by an asymmetric 8-base-pair core sequence. Recombination between loxP sites (i) can occur either inter-or intramolecularly, (ii) can occur when the sites are present on either supercoiled or linear DNA, and (iii) is independent of the re...

Mice deficient in hepatocyte nuclear factor 1 alpha (HNF-1␣) were produced by use of the Cre-loxP recombination system. HNF-1␣-null mice are viable but sterile and exhibit a phenotype reminiscent of both Laron-type dwarfism and non-insulin-dependent diabetes mellitus (NIDDM). In contrast to an earlier HNF-1␣-null mouse line that had been produced by use of standard gene disruption methodology (M. Pontoglio, J. Barra, M. Hadchouel, A. Doyen, C. Kress, J. P. Bach, C. Babinet, and M. Yaniv, Cell 84:575-585, 1996), these mice exhibited no increased mortality and only minimal renal dysfunction during the first 6 months of development. Both dwarfism and NIDDM are most likely due to the loss of expression of insulin-like growth factor I (IGF-I) and lower levels of insulin, resulting in stunted growth and elevated serum glucose levels, respectively. These results confirm the functional significance of the HNF-1␣ regulatory elements that had previously been shown to reside in the promoter regions of both the IGF-I and the insulin genes.