Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance for the efficiency of BAC/PAC recombineering techniques.

Cre is widely used for DNA tailoring and, in combination with recombineering techniques, to modify BAC/PAC sequences for generating transgenic animals. However, mammalian genomes contain recombinase recognition sites (cryptic loxP sites) that can promote illegitimate DNA recombination and damage when cells express the Cre recombinase gene. We have created a new bioinformatic tool, FuzznucComparator, which searches for cryptic loxP sites and we have applied it to the analysis of the whole mouse genome. We found that cryptic loxP sites occur frequently and are homogeneously distributed in the genome. Given the mammalian nature of BAC/PAC genomic inserts, we hypothesised that the presence of cryptic loxP sites may affect the ability to grow and modify BAC and PAC clones in E. coli expressing Cre recombinase. We have observed a defect in bacterial growth when some BACs and PACs were transformed into EL350, a DH10B-derived bacterial strain that expresses Cre recombinase under the control of an arabinose-inducible promoter. In this study, we have demonstrated that Cre recombinase expression is leaky in un-induced EL350 cells and that some BAC/PAC sequences contain cryptic loxP sites, which are active and mediate the introduction of single-strand nicks in BAC/PAC genomic inserts.

Granulation rescue and developmental marking of juxtaglomerular cells using "piggy-BAC" recombination of the mouse ren locus.

Mice lacking a functional Ren-1(d) gene exhibit a complete lack of renal juxtaglomerular cell granulation and atypical macula densa morphology. Transgenic mice carrying a 145-kilobase BAC clone encompassing the Ren-1(d) and Ren-2 loci were generated, characterized, and backcrossed with Ren-1(d-/-) mice. Homozygous Ren-1(d)-null mice expressing the BAC clone exhibited complete restoration of normal renal structure. Homologous recombination in Escherichia coli was used to generate a modified version of the BAC clone, in which an IRESbeta-geo cassette was inserted specifically into the Ren-1(d) gene. When introduced into the germline, the modified clone provided a marker for juxtaglomerular cell differentiation and beta-geo was expressed appropriately in juxtaglomerular cells throughout development. Parallel backcross experiments onto the Ren-1(d)-null background demonstrated that the juxtaglomerular cells expressed the modified Ren-1(d) locus in the absence of regranulation. These data demonstrate that the nongranulated cells constitute bona fide juxtaglomerular cells despite their altered morphology, that overexpression of renin-2 cannot compensate for the loss of renin-1(d), and that primary structural differences between the two isoforms are responsible for the differences in granulation. The use of BAC modification as part of functional complementation studies illustrates the potential for in vivo molecular dissection of key physiological mechanisms.

Efficient Cre-lox linearisation of BACs: applications to physical mapping and generation of transgenic animals.

Due to the size of BAC, PAC and P1 clones, it is often difficult to construct detailed restriction maps, with large number of restriction fragments leading to ambiguity of mapping data. We report the use of Cre recombinase to linearise and asymmetrically introduce label at the unique loxP site of large loxP-containing clones. Subsequent partial digestion allows the direct ordering of restriction fragments. Additionally, BAC DNA linearised using the Cre-lox system has been used successfully to generate transgenic animals.

Identification of Grf1 on mouse chromosome 9 as an imprinted gene by RLGS-M.

Normal mammalian development requires a diploid combination of both haploid parental genomes. Uniparental disomy for certain segments of specific chromosomes results in aberrant development or prenatal lethality, indicating that the parental genomes have undergone modifications during gametogenesis. These modifications result in parent-of-origin specific expression for some genes, a phenomenon called genomic imprinting. Recent work with DNA methyltransferase deficient mice showed that differential methylation is the probable basis of the imprinted character of several genes. Screening for endogenous imprinted loci using restriction landmark genomic scanning with methylation sensitive enzymes (RLGS-M) identified eight imprinted RLGS (Irigs) candidate loci. Molecular analysis of the genomic region of one of the loci (Irigs2) resulted in the discovery of the paternally imprinted U2afbp-rs gene within a previously identified imprinted region on mouse chromosome 11 (refs 5, 7). This paper describes the characterisation of a novel imprinted RLGS-M locus, Irigs3, on mouse chromosome 9 (ref. 6). Within this locus we identified the Grf1 (also called Cdc25Mm) gene, which is homologous to the RAS-specific guanine nucleotide exchange factor gene, CDC25, in Saccharomyces cerevisiae. Grf1 is located about 30 kb downstream of the methylation imprinted site, identified by RLGS-M, and shows paternal allele specific expression in mouse brain, stomach and heart. Our results indicate that imprinting may have a role in regulating mitogenic signal transduction pathways during growth and development.

[Oxidation state of membrane phospholipids and activity of the microsomal system of cholesterol hydroxylation in the liver of animals during atherogenesis]. / Stepen'okislennosti membrannykh fosfolipidov i aktivnost' mikrosomal'noi sistemy gidroksilirovaniia kholesterina v pecheni zhivotnykh pri aterogeneze.

Content of cholesterol in various tissues of experimental animals /blood plasma, thoracal department of aorta, liver microsomes/ was increased during atherogenesis depending on the level of aorta impairment. In atherogenesis content of both primary and secondary products of lipid peroxidation was also increased in microsomal membranes of rabbit and mini-pig liver tissue; the increase in the rate of microsomal lipid oxidation was accompanied by a decrease in the activity of membrane-bound microsomal 7 alpha-hydroxylase of cholesterol.

[Lipid peroxides and atherosclerosis. Free radical peroxidation of polyene lipids in the blood in ischemic heart disease]. / Perekisi lipidov i ateroskleroz, Svobodnoradikal'noe perekisnoe okislenie polienovykh lipidov v krovi bol'nykh ishemicheskoi bolezn'iu serdtsa.

Examination of blood in 114 males (35--60 years of age) with stage III of lipid hydroperoxides, acylhydroperoxides, intermol ecular cross-links in the aminophosphatides and secondary products of lipoperoxidation was increased considerably as compared to that in practically healthy males (30) of the same age. The activity of blood glutathione lipoperoxidase in the examined group of patients was sharply reduced.

[Lipid peroxides and atherosclerosis. The content of lipid peroxidation products in the blood in ischemic heart disease]. / Perekisi lipidov i ateroskleroz. Soderzhanie produktov perekisnogo okisleniia lipidov v krovi bol'nykh ishemicheskoi bolezni'iu serdtsa.

It was established that the content of primary (acylhydroperoxide) and secondary (intermollecular "seams" in aminophospholipids) products of lipid peroxide oxidation in blood of patients with ischemic heart disease is increased against the background of hyperlipidemia and hypercholesterolemia. It is suggested that intensification of lipid peroxide oxidation may play a role in the pathogenesis of atherosclerosis.

[Free radical peroxidation of liver mitochondrial and microsomal phospholipids in rat postnatal development]. / Svobodnoradikal'noe perekisnoe okislenie mitokhondrial'nykh i mikrosomal'nykh fosfolipidov pecheni v protsesse postnatal'nogo razvitiia krys.

Activities of both non-enzymic (ascorbate-dependent) and enzymic NADPH-dependent) peroxidation of microsomal and mitochondrial phospholipids are found to be increased in rat liver during postnatal development. It is suggested, that microsomal NADPH-dependent phospholipid dioxygenase forming a chemical modification of membrane polyenic acyls, can be a factor regulating the activities of membrane-linked enzymes under normal physiological processes.

[Increase in the lipid peroxide content in the blood and aortas of rabbits with experimental atherosclerosis]. / Uvelichenie soderzhaniia perekisei lipidov v krovi i aortakh krolikov s éksperimental'nym aterosklerozom

Content of lipid peroxides was estimated in blood and aorta of intact rabbits by means of highly sensitive polarographic method, using a mercuric electrode. Content of lipoperoxides in blood and aorta was increased 4.4- and 3.6-fold, respectively, in animals with pronounced experimental atherosclerosis. The oxidation of lipoperoxides caused a distinct increase in content of intermolecular bindings in amino-containing phospholipids in aorta of rabbits with pronounced atheromatosis.

[Role of structural factor in the kinetics of free-radical oxidation of lipids in the membranes]. / O roli strukturnogo faktora v kinetike svobodnoradikal'nogo okisleniia lipidov v membranakh

Kinetics of free radical oxidation of lipids (formation of primary molecular products - hydroperoxides, secondary products - carbonyl substances and "fluorescent pigments") was studied in various membrane fragments, which were distinctly differentiated by their fatty acid composition. The non-enzymatic catalysis of lipoperoxidation in these systems was initiated by addition of Fe2+ + ascorbic acid. The membrane fragments were isolated from microsomes and mitochondria of rat liver tissue, external segments of frog retina rods, sarcoplasmic reticulum of rabbit and carp skeletal muscles and from microsomes of rabbit brain. In membrane structures the free radical oxidation of lipids developed following the same pattern as the liquid-phase oxidation of alkenes. But in phospholipids of biomembranes the rate of hydroperoxides formation did not correlate with the level of unsaturation of their polyene acyls. The rate of peroxidation of unsaturated fatty acids was distinctly determined by their structural organization in membranes.

[Role of lipid peroxides in the pathogenesis of arteriosclerosis. Detoxication of lipid peroxides by the glutathione-peroxidase system in the aorta]. / Rol' perekisei lipidov v patogenese ateroskleroza. Detoksikatsiia lipoperekisei glutation-peroksidaznoi sistemoi v aorte

In aorta of intact rabbits the high activity of glutathione-peroxidase, which detoxicates lipoperoxides, was observed. In aorta of animals with pronounced experimental atheromatosis the enzyme activity did not distinctly differ from the control values. The animals with high initial content of glutathione-peroxidase in aorta were shown to be less subjected to the impairment in alimentary atherosclerosis.

[Lipid peroxides and arteriosclerosis]. / Perekisi lipidov i ateroskleroz

The significance of free radical oxidation of phospholipids in tissues of animals with experimental atherosclerosis was investigated. By using modern physico-chemical methods an elevated content of polyunsaturated fatty acids and other lipids peroxides was discovered in the blood and the aorta of rabbits with experimental atheromatosis. The human blood demonstrated a low level of protective enzymatic systems and a high content of products secondary to peroxidal oxidation of the lipids. The mechanism accounting for the action of lipids peroxides on the vascular wall resulting in the formation of atheromatous plaques is considered.