Site-specific Recombination Technology Research Articles

Even a low dose of tamoxifen profoundly induces adipose tissue browning in female mice.

Background:Tamoxifen-inducible Cre/lox site-specific recombination technology has been widely used to generate conditional transgenic mice. As an estrogen receptor ligand, tamoxifen itself potentially affects energy metabolism, which may confound interpretation of data especially in metabolic studies. Considering sexual dimorphism, in this study, the effects of low-dose tamoxifen administration on energy metabolism, and browning of adipose tissues in female and male mice were investigated.Methods:Female and male C57/BL6 mice were injected with tamoxifen oil solution (i.p.) and then housed at both room temperature (23 ± 2 °C) and cold environment (6 ± 1 °C). Serum, brown and white adipose tissues were obtained, and the effects of tamoxifen administration on energy metabolism and the browning of adipose tissues were evaluated.Results:At 25 mg/kg body weight (BDW), tamoxifen administration for 3 alternative days decreased the percentage of inguinal and gonadal white adipose tissue weights in female mice accompanied by the up-regulation of thermogenesis in adipose tissues. In contrast, this dosage of tamoxifen did not induce noticeable changes in the energy metabolism and thermogenesis of adipose tissue in male mice under room temperature. Consistently, under cold stimulus, substantial browning of adipose tissues was observed in female mice injected with tamoxifen (50 mg/kg BDW, single injection) but not in male mice. Two-way ANOVA tests also demonstrated significant interactions between tamoxifen treatment and gender on the expression of thermogenic markers in adipose tissues.Conclusion:Tamoxifen, even at a low dose, remarkably increases thermogenesis in adipose tissues of female mice; meanwhile, such a low dose could be used in male mice for inducing gene recombination without confounding the interpretation of data related to metabolism and thermogenesis of adipose tissues.

Development of marker-free transformants by site-specific recombinases

To produce transgenic plants in current biotechnology selectable marker genes are used that lead to the selectivity.of transformants from non-transformed organisms. However, after the transgenic event has been occurred, the presence of these genes in transformed genome in general is uselless. Moreover, the continued presence of this kind of genes in transgenic plants with their further commercialization may raise certain public concern. Therefore, various techniques have been developed in recent years to obtain marker free transgenic plants. In the present review the main strategies for removal of selective marker DNA sequences that are used in genetic engineering are described. The most popular among them is site-specific recombination technology. The particular attention is paid to site-specific recombinase system Cre/loxP. The using of a new approach with site-specific recombinase system Cre/loxP under the control of 35S promoter to generate marker-free genetically modified plants is described.

Site-specific recombination in the chicken genome using Flipase recombinase-mediated cassette exchange.

Targeted genome recombination has been applied in diverse research fields and has a wide range of possible applications. In particular, the discovery of specific loci in the genome that support robust and ubiquitous expression of integrated genes and the development of genome-editing technology have facilitated rapid advances in various scientific areas. In this study, we produced transgenic (TG) chickens that can induce recombinase-mediated gene cassette exchange (RMCE), one of the site-specific recombination technologies, and confirmed RMCE in TG chicken-derived cells. As a result, we established TG chicken lines that have, Flipase (Flp) recognition target (FRT) pairs in the chicken genome, mediated by piggyBac transposition. The transgene integration patterns were diverse in each TG chicken line, and the integration diversity resulted in diverse levels of expression of exogenous genes in each tissue of the TG chickens. In addition, the replaced gene cassette was expressed successfully and maintained by RMCE in the FRT predominant loci of TG chicken-derived cells. These results indicate that targeted genome recombination technology with RMCE could be adaptable to TG chicken models and that the technology would be applicable to specific gene regulation by cis-element insertion and customized expression of functional proteins at predicted levels without epigenetic influence.

Fitness cost implications of PhiC31-mediated site-specific integrations in target-site strains of the Mexican fruit fly, Anastrepha ludens (Diptera: Tephritidae).

Site-specific recombination technologies are powerful new tools for the manipulation of genomic DNA in insects that can improve transgenesis strategies such as targeting transgene insertions, allowing transgene cassette exchange and DNA mobilization for transgene stabilization. However, understanding the fitness cost implications of these manipulations for transgenic strain applications is critical. In this study independent piggyBac-mediated attP target-sites marked with DsRed were created in several genomic positions in the Mexican fruit fly, Anastrepha ludens. Two of these strains, one having an autosomal (attP_F7) and the other a Y-linked (attP_2-M6y) integration, exhibited fitness parameters (dynamic demography and sexual competitiveness) similar to wild type flies. These strains were thus selected for targeted insertion using, for the first time in mexfly, the phiC31-integrase recombination system to insert an additional EGFP-marked transgene to determine its effect on host strain fitness. Fitness tests showed that the integration event in the int_2-M6y recombinant strain had no significant effect, while the int_F7 recombinant strain exhibited significantly lower fitness relative to the original attP_F7 target-site host strain. These results indicate that while targeted transgene integrations can be achieved without an additional fitness cost, at some genomic positions insertion of additional DNA into a previously integrated transgene can have a significant negative effect. Thus, for targeted transgene insertions fitness costs must be evaluated both previous to and subsequent to new site-specific insertions in the target-site strain.

A New Resource for CharacterizingX-Linked Genes inDrosophila melanogaster: Systematic Coverage and Subdivision of theXChromosome With Nested,Y-Linked Duplications

Interchromosomal duplications are especially important for the study of X-linked genes. Males inheriting a mutation in a vital X-linked gene cannot survive unless there is a wild-type copy of the gene duplicated elsewhere in the genome. Rescuing the lethality of an X-linked mutation with a duplication allows the mutation to be used experimentally in complementation tests and other genetic crosses and it maps the mutated gene to a defined chromosomal region. Duplications can also be used to screen for dosage-dependent enhancers and suppressors of mutant phenotypes as a way to identify genes involved in the same biological process. We describe an ongoing project in Drosophila melanogaster to generate comprehensive coverage and extensive breakpoint subdivision of the X chromosome with megabase-scale X segments borne on Y chromosomes. The in vivo method involves the creation of X inversions on attached-XY chromosomes by FLP-FRT site-specific recombination technology followed by irradiation to induce large internal X deletions. The resulting chromosomes consist of the X tip, a medial X segment placed near the tip by an inversion, and a full Y. A nested set of medial duplicated segments is derived from each inversion precursor. We have constructed a set of inversions on attached-XY chromosomes that enable us to isolate nested duplicated segments from all X regions. To date, our screens have provided a minimum of 78% X coverage with duplication breakpoints spaced a median of nine genes apart. These duplication chromosomes will be valuable resources for rescuing and mapping X-linked mutations and identifying dosage-dependent modifiers of mutant phenotypes.

Recombinase technology: applications and possibilities

The use of recombinases for genomic engineering is no longer a new technology. In fact, this technology has entered its third decade since the initial discovery that recombinases function in heterologous systems (Sauer in Mol Cell Biol 7(6):2087–2096, 1987). The random insertion of a transgene into a plant genome by traditional methods generates unpredictable expression patterns. This feature of transgenesis makes screening for functional lines with predictable expression labor intensive and time consuming. Furthermore, an antibiotic resistance gene is often left in the final product and the potential escape of such resistance markers into the environment and their potential consumption raises consumer concern. The use of site-specific recombination technology in plant genome manipulation has been demonstrated to effectively resolve complex transgene insertions to single copy, remove unwanted DNA, and precisely insert DNA into known genomic target sites. Recombinases have also been demonstrated capable of site-specific recombination within non-nuclear targets, such as the plastid genome of tobacco. Here, we review multiple uses of site-specific recombination and their application toward plant genomic engineering. We also provide alternative strategies for the combined use of multiple site-specific recombinase systems for genome engineering to precisely insert transgenes into a pre-determined locus, and removal of unwanted selectable marker genes.

An in vivo imaging-based assay for detecting protein interactions over a wide range of binding affinities

Identifying and characterizing protein interactions are fundamental steps toward understanding and modeling biological networks. Methods that detect protein interactions in intact cells rather than buffered solutions are likely more relevant to natural systems since molecular crowding events in the cytosol can influence the diffusion and reactivity of individual proteins. One in vivo, imaging-based method relies on the colocalization of two proteins of interest fused to DivIVA, a cell division protein from Bacillus subtilis, and green fluorescent protein (GFP). We have modified this imaging-based assay to facilitate rapid cloning by constructing new vectors encoding N- and C-terminal DivIVA or GFP molecular tag fusions based on site-specific recombination technology. The sensitivity of the assay was defined using a well-characterized protein interaction system involving the eukaryotic nuclear import receptor subunit, Importin α (Impα), and variant nuclear localization signals (NLS) representing a range of binding affinities. These data demonstrate that the modified colocalization assay is sensitive enough to detect protein interactions with Kd values that span over four orders of magnitude (1nM to 15μM). Lastly, this assay was used to confirm numerous protein interactions identified from mass spectrometry-based analyses of affinity isolates as part of an interactome mapping project in Rhodopseudomonas palustris.

Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana

Site-specific recombination systems, such as Cre-lox from bacteriophage P1, have become very important tools for plant genome engineering. In many cases a constitutive promoter is used to express the recombinase gene. However, for certain research and commercial applications constitutive Cre-mediated recombination may not be desirable. We have evaluated the potential of seven different germline promoter:cre fusions to remove a stably integrated lox cassette through Cre-mediated recombination in Arabidopsis thaliana. We monitored the functionality of each promoter in the germline of primary transformants by analyzing the presence of the recombined lox cassette in T(2) progeny. The selected germline promoters are involved in different developmental cues, including early stem cell identity (CLAVATA3), flower meristem identity (LEAFY, APETALA1), floral organ identity (AGAMOUS), and meiosis (SOLO DANCERS, DMC1, SWITCH1). For five out of these seven promoters we were able to show that efficient Cre-mediated recombination does, indeed, occur and that the recombination takes place at some point during germline development. Furthermore, a recombination efficiency of 100% is obtained when Cre-expression is regulated by the CLAVATA3 promoter. In addition, with these promoters, we observe much less variation in recombination frequency than previously reported for the 35S promoter. For these reasons, we believe that germline-specific Cre-lox recombination provides an additional tool to the site-specific recombination technology in plants.

Application prospect of new site-specific recombination systems in gene transformation and gene stacking

Site-specific recombination technology provides an important means for regulating transgene expression and improving efficiency of gene transformation. This technique can lead to the translocation, inversion, deletion, and integration, and the expression of the affected genes can be manipulated in different tissues and organs at different developmental periods. Using site-specific recombination system in gene transformation, we can not only acquire plenty of transformants with precise structural fidelity and faithful expression, but also understand the function of new genes efficiently. Gene stacking technology based on the site-specific recombination can produce transgenic crops with good complex traits, which will accelerate the process of research and development of elite cultivars and provide technical support for the exploration of transgenic crops in China.

GM maize from site-specific recombination technology, what next?

The term plant genetic engineering has long conveyed a highly efficient and precise process for the manipulation of plant genomes. For nearly two decades, research on recombinase-based applications has steadily advanced the surgical capabilities of plant genome rearrangements. Once considered interesting laboratory exercises, a first crop plant derived from this type of DNA acrobatics is heading to market. Originally configured for a specific application, to remove a selectable marker, it could be the first of more to come - and not just market-free plants.

The variable number of tandem repeats element in DAT1 regulates in vitro dopamine transporter density

BackgroundA 40-bp variable number of tandem repeats (VNTR) polymorphism exists in the 15th exon of DAT1, the gene encoding the human dopamine transporter (DAT). Though the VNTR resides in a region encoding the 3' untranslated region (UTR) and does not alter the protein's amino acid sequence, the prevalent 10-repeat variant has shown both linkage and association to Attention Deficit Hyperactivity Disorder (ADHD). In this study, we examined the effects of the DAT1 VNTR on measures of in vitro DAT expression and pharmacology. A series of four DAT1 constructs, each containing the DAT1 coding region, but varying with respect to the downstream presence or content of the 3'UTR, were engineered and stably transfected into an HEK-293 variant using Flp-In integration, an enzyme-mediated, site-specific recombination technology.Results[3H] Win 35,428 saturation binding assays and DAT immunoblots revealed statistically significant differences in DAT expression attributable to DAT1 genotype. Cells harboring the 10-repeat DAT1 variant were characterized by a Bmax approximately 50% greater than cells with the 9-repeat VNTR; those containing only the DAT1 coding region or the coding region flanked by a truncated 3' UTR resulted in greater DAT density than either of the naturalistic 9- and 10-repeat variants. Competition binding assays showed no statistically significant DAT1 genotype effects on the DAT affinity for methylphenidate, a finding consistent with the positional location of the VNTR.ConclusionThis study identified the DAT1 VNTR as a functional polymorphism and provides an interpretive framework for its association with behavioral phenotypes.