Chimeric Cre Research Articles

Marker-free transgenic rice expressing the vegetative insecticidal protein (Vip) of Bacillus thuringiensis shows broad insecticidal properties.

Genetically engineered rice lines with broad insecticidal properties against major lepidopteran pests were generated using a synthetic, truncated form of vegetative insecticidal protein (Syn vip3BR) from Bacillus thuringiensis. The selectable marker gene and the redundant transgene(s) were eliminated through Cre/ lox mediated recombination and genetic segregation to make consumer friendly Bt -rice. For sustainable resistance against lepidopteran insect pests, chloroplast targeted synthetic version of bioactive core component of a vegetative insecticidal protein (Synvip3BR) of Bacillus thuringiensis was expressed in rice under the control of green-tissue specific ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit gene promoter. The transgenic plants (in Oryza sativa indica Swarna cultivar) showed high insect mortality rate in vitro against major rice pests, yellow stem borer (Scirpophaga incertulas), rice leaf folder (Cnaphalocrocis medinalis) and rice horn caterpillar (Melanitis leda ismene) in T1 generation, indicating insecticidal potency of Synvip3BR. Under field conditions, the T1 plants showed considerable resistance against leaf folders and stem borers. The expression cassette (vip-lox-hpt-lox) as well as another vector with chimeric cre recombinase gene under constitutive rice ubiquitin1 gene promoter was designed for the elimination of selectable marker hygromycin phosphotransferase (hptII) gene. Crossing experiments were performed between T1 plants with single insertion site of vip-lox-hpt-lox T-DNA and one T1 plant with moderate expression of cre recombinase with linked bialaphos resistance (synbar) gene. Marker gene excision was achieved in hybrids with up to 41.18% recombination efficiency. Insect resistant transgenic lines, devoid of selectable marker and redundant transgene(s) (hptII+cre-synbar), were established in subsequent generation through genetic segregation.

Tyk2 and Stat3 Regulate Brown Adipose Tissue Differentiation and Obesity

Mice lacking the Jak tyrosine kinase member Tyk2 become progressively obese due to aberrant development of Myf5+ brown adipose tissue (BAT). Tyk2 RNA levels in BAT and skeletal muscle, which shares a common progenitor with BAT, are dramatically decreased in mice placed on a high-fat diet and in obese humans. Expression of Tyk2 or the constitutively active form of the transcription factor Stat3 (CAStat3) restores differentiation in Tyk2(-/-) brown preadipocytes. Furthermore, Tyk2(-/-) mice expressing CAStat3 transgene in BAT also show improved BAT development, normal levels of insulin, and significantly lower body weights. Stat3 binds to PRDM16, a master regulator of BAT differentiation, and enhances the stability of PRDM16 protein. These results define Tyk2 and Stat3 as critical determinants of brown fat lineage and suggest that altered levels of Tyk2 are associated with obesity in both rodents and humans.

Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting

BackgroundThe use of the Cre/loxP system for gene targeting has been proven to be a powerful tool for understanding gene function. The purpose of this study was to create and characterize an inducible, skeletal muscle-specific Cre transgenic mouse strain.MethodsTo achieve skeletal muscle-specific expression, the human α-skeletal actin promoter was used to drive expression of a chimeric Cre recombinase containing two mutated estrogen receptor ligand-binding domains.ResultsWestern blot analysis, PCR and β-galactosidase staining confirmed that Cre-mediated recombination was restricted to limb and craniofacial skeletal muscles only after tamoxifen administration.ConclusionsA transgenic mouse was created that allows inducible, gene targeting of floxed genes in adult skeletal muscle of different developmental origins. This new mouse will be of great utility to the skeletal muscle community.

Selectable antibiotic resistance marker gene-free transgenic rice harbouring the garlic leaf lectin gene exhibits resistance to sap-sucking planthoppers

Rice, the major food crop of world is severely affected by homopteran sucking pests. We introduced coding sequence of Allium sativum leaf agglutinin, ASAL, in rice cultivar IR64 to develop sustainable resistance against sap-sucking planthoppers as well as eliminated the selectable antibiotic-resistant marker gene hygromycin phosphotransferase (hpt) exploiting cre/lox site-specific recombination system. An expression vector was constructed containing the coding sequence of ASAL, a potent controlling agent against green leafhoppers (GLH, Nephotettix virescens) and brown planthopper (BPH, Nilaparvata lugens). The selectable marker (hpt) gene cassette was cloned within two lox sites of the same vector. Alongside, another vector was developed with chimeric cre recombinase gene cassette. Reciprocal crosses were performed between three single-copy T(0) plants with ASAL- lox-hpt-lox T-DNA and three single-copy T(0) plants with cre-bar T-DNA. Marker gene excisions were detected in T(1) hybrids through hygromycin sensitivity assay. Molecular analysis of T(1) plants exhibited 27.4% recombination efficiency. T(2) progenies of L03C04(1) hybrid parent showed 25% cre negative ASAL-expressing plants. Northern blot, western blot and ELISA showed significant level of ASAL expression in five marker-free T(2) progeny plants. In planta bioassay of GLH and BPH performed on these T(2) progenies exhibited radical reduction in survivability and fecundity compared with the untransformed control plants.

Defining the hair follicle stem cell (Part I)

Defining the hair follicle stem cell (Part I)

A chimeric Cre recombinase with regulated directionality

From bacterial viruses to humans, site-specific recombination and transposition are the major pathways for rearranging genomes on both long- and short-time scales. The site-specific pathways can be divided into 2 groups based on whether they are stochastic or regulated. Recombinases Cre and lambda Int are well-studied examples of each group, respectively. Both have been widely exploited as powerful and flexible tools for genetic engineering: Cre primarily in vivo and lambda Int primarily in vitro. Although Cre and Int use the same mechanism of DNA strand exchange, their respective reaction pathways are very different. Cre-mediated recombination is bidirectional, unregulated, does not require accessory proteins, and has a minimal symmetric DNA target. We show that when Cre is fused to the small N-terminal domain of Int, the resulting chimeric Cre recombines complex higher-order DNA targets comprising >200 bp encoding 16 protein-binding sites. This recombination requires the IHF protein, is unidirectional, and is regulated by the relative levels of the 3 accessory proteins, IHF, Xis, and Fis. In one direction, recombination depends on the Xis protein, and in the other direction it is inhibited by Xis. It is striking that regulated directionality and complexity can be conferred in a simple chimeric construction. We suggest that the relative ease of constructing a chimeric Cre with these properties may simulate the evolutionary interconversions responsible for the large variety of site-specific recombinases observed in Archaea, Eubacteria, and Eukarya.

Cre/lox system to develop selectable marker free transgenic tobacco plants conferring resistance against sap sucking homopteran insect

A binary expression vector was constructed containing the insecticidal gene Allium sativum leaf agglutinin (ASAL), and a selectable nptII marker gene cassette, flanked by lox sites. Similarly, another binary vector was developed with the chimeric cre gene construct. Transformed tobacco plants were generated with these two independent vectors. Each of the T(0) lox plants was crossed with T(0) Cre plants. PCR analyses followed by the sequencing of the target T-DNA part of the hybrid T(1) plants demonstrated the excision of the nptII gene in highly precised manner in certain percentage of the T(1) hybrid lines. The frequency of such marker gene excision was calculated to be 19.2% in the hybrids. Marker free plants were able to express ASAL efficiently and reduce the survivability of Myzus persiceae, the deadly pest of tobacco significantly, compared to the control tobacco plants. Results of PCR and Southern blot analyses of some of the T(2) plants detected the absence of cre as well as nptII genes. Thus, the crossing strategy involving Cre/lox system for the excision of marker genes appears to be very effective and easy to execute. Documentation of such marker excision phenomenon in the transgenic plants expressing the important insecticidal protein for the first time has a great significance from agricultural and biotechnological points of view.

Disturbed Epidermal Structure in Mice with Temporally Controlled Fatp4 Deficiency

So far, little is known about the physiological role of fatty acid transport protein 4 (Fatp4, Slc27a4). Mice with a targeted disruption of the Fatp4 gene display features of a human neonatally lethal restrictive dermopathy with a hyperproliferative hyperkeratosis, a disturbed epidermal barrier, a flat dermal-epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis, demonstrating that Fatp4 is necessary for the formation of the epidermal barrier. Because Fatp4 is widely expressed, it is unclear whether intrinsic Fatp4 deficiency in the epidermis alone can cause changes in the epidermal structure or whether the abnormalities observed are secondary to the loss of Fatp4 in other organs. To evaluate the functional role of Fatp4 in the skin, we generated a mouse line with Fatp4 deficiency inducible in the epidermis. Mice with epidermal keratinocyte-specific Fatp4 deficiency developed a hyperproliferative hyperkeratosis with a disturbed epidermal barrier. These changes resemble the histological abnormalities in the epidermis of newborn mice with total Fatp4 deficiency. We conclude that Fatp4 in epidermal keratinocytes is essential for the maintenance of a normal epidermal structure.

Genetic Evidence for an Interaction between a Picornaviral cis-Acting RNA Replication Element and 3CD Protein

Internally located, cis-acting RNA replication elements, termed cres, are essential for replication of the genomes of picornaviruses such as human rhinovirus 14 (HRV-14) and poliovirus because they template uridylylation of the protein primer, VPg, by the polymerase 3D(pol). These cres form stem-loop structures sharing a common loop motif, and the HRV-14 cre can substitute functionally for the poliovirus cre in both uridylylation in vitro and RNA replication in vivo. We show, however, that the poliovirus cre is unable to support HRV-14 RNA replication. This lack of complementation maps to the stem of the poliovirus cre and was reversed by single nucleotide substitutions in the stem as well as the base of the loop. Replication-competent, revertant viruses rescued from dicistronic HRV-14 RNAs containing the poliovirus cre, or a chimeric cre containing the poliovirus stem, contained adaptive amino acid substitutions. These mapped to the surface of both the polymerase 3D(pol), at the tip of the "thumb" domain, and the protease 3C(pro), on the side opposing the active site and near the end of an extended strand segment implicated previously in RNA binding. These mutations substantially enhanced replication competence when introduced into HRV-14 RNAs containing the poliovirus cre, and they were additive in their effects. The data support a model in which 3CD or its derivatives 3C(pro) and 3D(pol) interact directly with the stem of the cre during uridylylation of VPg.

A tamoxifen-inducible chimeric Cre recombinase specifically effective in the fetal and adult mouse liver

The spatiotemporal control of somatic mutagenesis in mice is considered a promising step to determine the function of a given gene product in a defined population of cells at any given time during animal life and also to generate better mouse models of human diseases. To introduce defined mutations in a temporally controlled manner in the liver, we established transgenic mice expressing a tamoxifen-inducible Cre recombinase under the control of the transthyretin promoter ( TTR-Cre ind). The recombinase activity was examined on 2 different floxed alleles by crossing TTR-Cre ind mice with either the reporter strain ROSA 26 or with homozygous mice carrying floxed catalytic α2 subunit of the adenosine monophosphate (AMP)-activated protein kinase gene. By placing 2 mutated hormone-binding domains of murine estrogen receptor (Mer) at both termini of the Cre, we show that the fusion protein is active only on administration of the synthetic estrogen antagonist 4-hydroxytamoxifen (4-OHT) without any background in the absence of the inducing agent. The recombination is specific of the fetal and adult liver, and we show that the efficiency of recombination reached 80% to 100% after treatment with 4-OHT. In conclusion, TTR-Cre ind transgenic mice represent a valuable tool for temporally controlling the desired gene modifications in vivo in the fetal and adult liver. This would certainly help to understand the physiologic functions of genes in the liver, to create various mouse models mimicking human diseases, and to contribute to liver cancer–specific suicide gene therapy studies. (H EPATOLOGY 2002;35:1072-1081.)

Selective ablation of retinoid X receptor α in hepatocytes impairs their lifespan and regenerative capacity

Retinoid X receptors (RXRs) are involved in a number of signaling pathways as heterodimeric partners of numerous nuclear receptors. Hepatocytes express high levels of the RXRalpha isotype, as well as several of its putative heterodimeric partners. Germ-line disruption (knockout) of RXRalpha has been shown to be lethal in utero, thus precluding analysis of its function at later life stages. Hepatocyte-specific disruption of RXRalpha during liver organogenesis has recently revealed that the presence of hepatocytes is not mandatory for the mouse, at least under normal mouse facility conditions, even though a number of metabolic events are impaired [Wan, Y.-J., et al. (2000) Mol. Cell. Biol. 20, 4436-4444]. However, it is unknown whether RXRalpha plays a role in the control of hepatocyte proliferation and lifespan. Here, we report a detailed analysis of the liver of mice in which RXRalpha was selectively ablated in adult hepatocytes by using the tamoxifen-inducible chimeric Cre recombinase system. Our results show that the lifespan of adult hepatocytes lacking RXRalpha is shorter than that of their wild-type counterparts, whereas proliferative hepatocytes of regenerating liver exhibit an even shorter lifespan. These lifespan shortenings are accompanied by increased polyploidy and multinuclearity. We conclude that RXRalpha plays important cell-autonomous function(s) in the mechanism(s) involved in the lifespan of hepatocytes and liver regeneration.

Inducible gene targeting in postnatal myocardium by cardiac-specific expression of a hormone-activated Cre fusion protein.

Cardiac-restricted expression of Cre recombinase can provoke lineage-specific gene excision in the myocardium. However, confounding early lethality may still preclude using loss-of-function models to study the postnatal heart. Here, we have tested whether inducible, heart-specific recombination can be triggered after birth by transgenic expression of a Cre fusion protein that incorporates a mutated progesterone receptor ligand binding domain (PR1) that is activated by the synthetic antiprogestin, RU486, but not by endogenous steroid hormones. CrePR1 driven by the alpha-myosin heavy chain (alphaMHC) promoter was expressed specifically in heart. Translocation of CrePR1 from cytoplasm to nuclei in ventricular myocytes was induced by RU486. To establish whether this approach can mediate cardiac-specific, drug-dependent excision between loxP sites in vivo, we mated alphaMHC-CrePR1 mice with a ubiquitously expressed (ROSA26) Cre reporter line. Offspring harboring alphaMHC-CrePR1 and/or the floxed allele were injected with RU486 versus vehicle, and the prevalence of beta-galactosidase (beta-gal)-positive cells was determined, indicative of Cre-mediated excision. Little or no baseline recombination was seen 1 week after birth. Cardiac-restricted, RU486-inducible recombination was demonstrated in bigenic mice at age 3 and 6 weeks, using each of 3 independent CrePR1 lines. Recombination in the absence of ligand paralleled the levels of CrePR1 protein expression and was more evident at 6 weeks. Thus, conditional, posttranslational activation of a Cre fusion protein can bypass potential embryonic and perinatal effects on the heart and permits inducible recombination in cardiac muscle. High levels of the chimeric Cre protein, in particular, were associated with progressive recombination in the absence of drug.

Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes

Retinoid X receptor alpha (RXRalpha) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXRalpha gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-ERT2 recombination system. Mice lacking RXRalpha in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXRalpha is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXRalpha in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor gamma.

Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha mediated by a tamoxifen-inducible chimeric Cre recombinase (Cre-ERT2) in adipocytes.

Retinoid X receptor alpha (RXRalpha) is involved in multiple signaling pathways, as a heterodimeric partner of several nuclear receptors. To investigate its function in energy homeostasis, we have selectively ablated the RXRalpha gene in adipocytes of 4-week-old transgenic mice by using the tamoxifen-inducible Cre-ERT2 recombination system. Mice lacking RXRalpha in adipocytes were resistant to dietary and chemically induced obesity and impaired in fasting-induced lipolysis. Our results also indicate that RXRalpha is involved in adipocyte differentiation. Thus, our data demonstrate the feasibility of adipocyte-selective temporally controlled gene engineering and reveal a central role of RXRalpha in adipogenesis, probably as a heterodimeric partner for peroxisome proliferator-activated receptor gamma.

Regulation of Cre Recombinase Activity by Mutated Estrogen Receptor Ligand-Binding Domains

Ligand-dependent chimeric Cre recombinases are powerful tools to induce specific DNA rearrangements in cultured cells and in mice. We report here the construction and characterization of a series of chimeric recombinases, each consisting of Cre fused to a mutated human oestrogen receptor (ER) ligand-binding domain (LBD). Two new ligand-dependent recombinases which contain either the G400V/M543A/L544A or the G400V/L539A/L540A triple mutation of the human ER LBD are efficiently induced by the synthetic ER antagonists 4-hydroxytamoxifen (OHT) and ICI 182,780 (ICI), respectively, but are insensitive to 17β-oestradiol (E2). Both chimeric recombinases should be useful for efficient spatio-temporally controlled site-directed somatic mutagenesis.

Site-directed recombination in the genome of transgenic tobacco

The plant genome responds to the bacteriophage P1-derived loxP-Cre site-specific recombination system. Recombination took place at loxP sites stably integrated in the tobacco genome, indicating that the Cre recombinase protein, expressed by a chimeric gene also stably resident in the genome, was able to enter the nucleus and to locate a specific 34 bp DNA sequence. An excisional recombination event was monitored by the acquisition of kanamycin resistance, which resulted from the loss of a polyadenylation signal sequence that interrupted a chimeric neomycin phosphotransferase II gene. Molecular analysis confirmed that the excision had occurred. Recombination occurred when plants with the integrated loxP construction were stably re-transformed with a chimeric cre gene and when plants with the introduced loxP construction were cross-bred with those carrying the chimeric cre gene. As assayed phenotypically, site-specific recombination could be detected in 50%-100% of the plants containing both elements of the system. Kanamycin resistance was detected at 2-3 weeks after re-transformation and in the first leaf of hybrid seedlings. This demonstration of the effectiveness of the loxP-Cre system in plants provides the basis for development of this system for such purposes as directing site-specific integration and regulation of gene expression.