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Construction of glucose-repressible yeast expression vectors.

A set of two episomal yeast expression vectors, pYME1 and pYME2, were constructed. These Saccharomyces cerevisiae-Escherichia coli shuttle vectors each contain a modified yeast MAL6S (encoding maltase) promoter that is expressed constitutively, but is subject to carbon catabolite repression by glucose. Expression from this promoter is still dependent upon the presence of active MALR (regulatory) protein. These expression vectors are particularly useful because most S. cerevisiae strains are MAL+, thereby exhibiting a wider host range than GAL-based vector systems. These pYME1 and pYME2 vectors are capable of expression to levels comparable to GAL-based expression plasmids and much higher than a variety of other repressible promoter vectors. The vectors are identical, except that their multiple cloning sites (MCS) are in opposite orientations, making them convenient for inserting heterologous genes.

Base Sequence↗

Lentivirus envelope sequences and proviral genomes are stabilized in Escherichia coli when cloned in low-copy-number plasmid vectors.

A promoter-selection vector (pKK232-8) was used to identify sequences with strong Escherichia coli promoter activity positioned near the start of the envelope-encoding genes (env) of two lentiviruses, simian immunodeficiency virus (SIV) and equine infectious anemia virus (EIAV). For EIAV, cloning the cryptic promoter sequences together with downstream sequences encoding the envelope glycoprotein (gp90) in moderate- to high-copy-number (hcn) plasmid vectors, such as pBR322 or pUC, resulted in rearrangements and point mutations in env when propagated in E. coli. To alleviate this problem, low-copy-number (lcn) cloning vectors, pLG338-30 and pLG339-SPORT, were constructed. The plasmids carry resistance markers for ampicillin (ApR) or kanamycin (KmR), the pSC101 origin of replication (ori) from plasmid pLG338 [Stoker et al., Gene 18 (1982) 335-341], and a multiple cloning site (MCS) from plasmids pIBI30 or pSPORT. Full-length env and genomic proviral sequences of EIAV were genetically stable when subcloned into these lcn vectors. Proviral sequences of an SIV clone (pBK28-SIV) that are genetically unstable in the hcn vector pUC18 were also stabilized and remained fully infectious when subcloned into the lcn vector pLG339-SPORT. These lcn vectors appear to be generally useful in stabilizing lentivirus genomic sequences for subcloning, propagation, and manipulation in E. coli, possibly as a result of reducing the level of toxic gene expression from cryptic promoter sequences.

Cloning, Molecular↗

The pCLIP plasmids: versatile cloning vectors based on the bacteriophage lambda origin of replication.

A series of general-purpose plasmid vectors based on the phage lambda origin of replication (ori) has been constructed. Each vector consists of a backbone plasmid encoding chloramphenicol resistance (CmR) and containing a unique HaeII site into which the lacZ alpha-complementing multiple cloning site (MCS) region of an established vector was inserted. To increase the cloning potential of the inserted MCS, superfluous restriction sites in the backbone were removed by a variety of techniques. The vectors, designated pCLIP (for CmR lambda ori integration proficient) plasmids, are of medium copy number and are compatible with most other vectors in common use. A total of 17 unique restriction sites in pCLIP8, pCLIP9, pCLIP18, pCLIP19 and pCLIP23 are available for cloning. As well as possessing the usual properties of vectors, the pCLIP plasmids are able to integrate reversibly into lambda prophage by homologous recombination. Thus, cloned DNA can be maintained in single or multiple copy at will. By integrating recombinant plasmids into appropriate deletion prophages followed by induction, phage::plasmid hybrids are produced which can be manipulated as phage. These properties are demonstrated using a test recombinant plasmid, pCLIPLEU2. The pCLIP vectors are therefore useful for routine plasmid cloning, complementation analysis and applications where the ability to manipulate recombinants in plasmid, phage or prophage forms is advantageous.

Amino Acid Sequence↗

An autonomously propagating luciferase-encoding vector for Dictyostelium discoideum.

We have constructed a luc reporter vector for Dictyostelium discoideum using a 626-bp fragment from the nuclear-associated plasmid Ddp2. The ori from Ddp2 is localized within this fragment and was used to provide an autonomous replication sequence for the reporter vector. This reporter vector was stably retained in D. discoideum AX3K cells without alteration. The vector molecule was also found to exist in relatively low copy number compared to other Dictyostelium vectors in the transformed cells. We demonstrated the utility of this vector as a reporter vector with glycogen synthase promoter/luc fusions of varying sizes.

Animals↗

P element transformation vectors for studying Drosophila melanogaster oogenesis and early embryogenesis.

We have constructed six new P-element-based Drosophila melanogaster transformation vectors that specifically allow for the high-level accumulation of any RNA of interest in the developing egg and pre-blastoderm embryo. Such specificity results, in part, from the inclusion in the vectors of an enhancer active exclusively in nurse cells, the principal providers of RNA to the egg and early embryo. The nurse cell enhancer was derived from the hsp26 heat-shock (HS) gene, but its activity was neither dependent on nor sensitive to HS. In addition to the nurse cell enhancer, two of the vectors contain sequences from the K10 gene that promote the early transfer of RNAs from nurse cells into the oocyte; RNAs that contain the K10 sequence are transferred into the oocyte during the early to middle stages of oogenesis (i.e., during stages 2-9), while RNAs that lack such sequences are stored in nurse cells until stage 11. All of the vectors contain a tsp and a multiple cloning site (MCS) immediately downstream from the hsp26 nurse cell enhancer. In three of the vectors, the MCS is preceded by an ATG start codon. A wild-type copy of the white gene is included in all of the vectors as a selectable marker for transformation. The specificity of the vectors was demonstrated by the analysis of the expression patterns of lacZ derivatives.

Amino Acid Sequence↗

Low- and high-copy-number shuttle vectors for replication in the budding yeast Kluyveromyces lactis.

Four sets of plasmid vectors for the budding yeast Kluyveromyces lactis (Kl) have been constructed. All plasmids are pUC19-based shuttle vectors having multiple unique sites in their multiple cloning site (MCS) within the bacterial lacZ gene. The first set of vectors contains Klori, the origin of replication for Kl isolated from Kluyveromyces plasmid pKD1, and one of the selectable nutritional markers, URA3, TRP1 or LEU2. These markers from the yeast, Saccharomyces cerevisiae (Sc), can complement the uraA1, trp1 and leu2 mutations of Kl. The second set of vectors, in addition to Klori, contains the ARS (autonomously replicating sequence) and centromeric sequences of Sc, and are able to replicate in both Sc and Kl. The third group of plasmids is centromeric vectors that are maintained in Kl at low copy number. The last family of vectors was designed for gene overexpression. As they contain the bacterial kanamycin-resistance-encoding gene (kan), plasmid copy number can be amplified to over 100 copies per cell in Kl by growing cells in the presence of the antibiotic G418 (Geneticin). This type of vector has been used to study the high-copy-lethality phenotype of a truncated version of the Kl MGI2 gene encoding the alpha-subunit of the mitochondrial F1F0-ATP synthase.

Cloning, Molecular↗

Comparative transduction efficiencies of human and nonhuman adenoviral vectors in human, murine, bovine, and porcine cells in culture.

Clinical usefulness of human Ad serotype 5 (HAd5) based vectors is limited primarily because of preexisting Ad immunity and lack of targeting to specific cell types. Alternative vectors based on less prevalent HAd serotypes as well as nonhuman adenoviruses such as porcine Ad serotype 3 (PAd3) and bovine Ad serotype 3 (BAd3) are being developed to overcome these shortcomings. Using virus neutralization assay, we examined whether preexisting Ad immunity in humans would cross-neutralize PAd3 or BAd3. To further evaluate the potential of PAd3 and BAd3 vectors as gene delivery vehicles, we compared their transduction efficiencies in a panel of human, murine, bovine, and porcine cell lines to those obtained with a HAd5 vector. Transduction by the HAd5 vector in the majority of human cell lines correlated with the expression levels of coxsackievirus-adenovirus receptor (CAR), the primary HAd5 receptor; while transduction by PAd3 and BAd3 vectors was CAR-independent. The results suggest that PAd3 and BAd3 vectors are promising gene delivery vehicles for human gene therapy as well as for recombinant vaccines for human and animal use.

Animals↗

Generation of variable and fixed length siRNA from a novel siRNA expression vector.

Small interfering RNA (siRNA) expression vectors using a Pol III promoter fall into two categories, vectors with a single Pol III promoter that express small hairpin RNA (shRNA) and vectors with two head-to-head (convergent) Pol III promoters that express siRNA. There are technical difficulties in preparing convergent siRNA vectors from cDNA. Here, we report construction of a novel convergent siRNA expression vector, pTHUB. Two XcmI sites were inserted between opposing Pol III promoters. After linearization with XcmI, pTHUB has a single 3' A overhang at each end that allows direct cloning of partially DNase I digested cDNA fragments (20-30 bp) tailed with ddT. A derivative method for generating 19 bp siRNA in pTHUB is also described. The suppression efficiency of the pTHUB vector is comparable to those of conventional shRNA vectors. We have made a siRNA library from a single cDNA. The same approach can be used to construct whole-genome siRNA libraries from cellular cDNA.

Base Sequence↗

Modified montmorillonite as vector for gene delivery.

Currently, gene delivery systems can be divided into two parts: viral or non-viral vectors. In general, viral vectors have a higher efficiency on gene delivery. However, they may sometimes provoke mutagenesis and carcinogenesis once re-activating in human body. Lots of non-viral vectors have been developed that tried to solve the problems happened on viral vectors. Unfortunately, most of non-viral vectors showed relatively lower transfection rate. The aim of this study is to develop a non-viral vector for gene delivery system. Montmorillonite (MMT) is one of clay minerals that consist of hydrated aluminum with Si-O tetrahedrons on the bottom of the layer and Al-O(OH)2 octahedrons on the top. The inter-layer space is about 12 A. The room is not enough to accommodate DNA for gene delivery. In the study, the cationic hexadecyltrimethylammonium (HDTMA) will be intercalated into the interlayer of MMT as a layer expander to expand the layer space for DNA accommodation. The optimal condition for the preparation of DNA-HDTMA-MMT is as follows: 1 mg of 1.5CEC HDTMA-MMT was prepared under pH value of 10.7 and with soaking time for 2 h. The DNA molecules can be protected from nuclease degradation, which can be proven by the electrophoresis analysis. DNA was successfully transfected into the nucleus of human dermal fibroblast and expressed enhanced green fluorescent protein (EGFP) gene with green fluorescence emission. The HDTMA-MMT has a great potential as a vector for gene delivery in the future.

Aluminum↗

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

Adenoviridae↗

Detection of osteoclastic cell-cell fusion through retroviral vector packaging.

Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.

Animals↗

Efficient transduction and seeding of human endothelial cells onto metallic stents using bicistronic pseudo-typed retroviral vectors encoding vascular endothelial growth factor.

BACKGROUND: Stents seeded with genetically modified endothelial cells (EC) may provide an attractive therapeutic modality for treating vascular diseases by combining the mechanical properties of the metallic stent with the biologic activity of native or genetically engineered ECs. The clinical feasibility of implanting seeded stents depends on the ability to achieve adequate stent coverage within a clinically applicable time frame. We tested the hypothesis that this goal could be achieved by seeding stents with human ECs overexpressing vascular endothelial growth factor (VEGF) and by using an efficient gene transfer system. METHODS AND RESULTS: Efficiency of gene transfer to human ECs using an amphotropic retroviral vector and a gibbon ape leukemia virus (GALV) pseudo-typed retroviral vector was examined and compared. For assessment of transduction rates, LacZ-encoding vectors were used and beta-galactosidase activity was determined 48 h after gene transfer. The transduction rate of primary human ECs using the amphotropic retroviral vector encoding the LacZ gene was low (2.9+/-2% of cells). Under the same conditions, the GALV pseudo-typed vector encoding LacZ transduced 94+/-2% of cells (P<.001). To test the effect of VEGF gene transfer on stent coverage, we transduced ECs using a bicistronic GALV pseudo-typed retroviral vector encoding either GFP alone or both VEGF and GFP. Since all transduced cells expressed GFP, stent coverage by ECs could be assessed by fluorescent inverted microscopy, which demonstrated that stent coverage by ECs overexpressing VEGF was more rapid and effective than coverage by ECs overexpressing GFP. Progressively increasing quantities of VEGF protein were detected in the conditioned medium of stents seeded with endothelia cells expressing VEGF 2, 3, and 5 days after seeding. CONCLUSIONS: High-rate gene transfer to human primary ECs was observed 48 h after transduction with GALV pseudo-typed retroviral vectors, eliminating the need for the time-consuming process of cell selection. Seeding with ECs overexpressing VEGF improved stent coverage and was associated with continuing secretion of the protein. The findings provide support for the feasibility of implanting genetically engineered biologically active cellular-coated stents.

Blood Vessel Prosthesis↗

Improved transduction of human corneal epithelial progenitor cells with cell-targeting adenoviral vectors.

The development of vectors and techniques to transfer therapeutic genes to corneal epithelium has broad clinical applications. To determine if adenoviral (Ad5) vectors could be tailored to increase transduction of corneal epithelial progenitor cells expressing epidermal growth factor receptor (EGFR), the feasibility of targeting gene therapy vectors to genetically modify primary cultured human corneal epithelial cells (PHCEC) was evaluated. PHCECs were cultured from human limbal explants and transduced with Ad5 vectors containing the enhanced green fluorescent protein (GFP) reporter cassette to mediate gene transfer. The efficiencies of transduction with different Ad5 dosages and different time periods of exposure were compared. Metabolically biotinylated Ad5 vectors were retargeted to PHCECs using biotinylated epidermal growth factor (EGF) as a cell-targeting ligand. Phenotypes and function assays of transduced cells were determined by real-time PCR and BrdU incorporation. Ad5 vectors transduced approximately 50-93% of PHCEC at 10-100 PFU/cell in a dose-dependent manner and the transgene persisted for more than 2 weeks in vitro. Retargeting of biotinylated Ad5 with EGF increased transduction of EGFR and ABCG2-expressing corneal epithelial progenitor cells up to nine-fold and reduced transduction of K12 and involucrin-expressing differentiated corneal epithelial cells and had higher BrdU incorporation indexes. These data provide proof of principle that ligand-bearing modified Ad5 vectors can target a population of corneal epithelial progenitor cells for corneal gene therapy.

Adenoviridae↗

A multipurpose transposon-based vector system mediates protein expression in Rhodococcus erythropolis.

In the current study we developed two transposon-based vectors; namely pTNR-KA and pTNR-TA and utilized them for expression of proteasome complex, derived from Streptomyces coelicolor, in Rhodococcus erythropolis. The two vectors can be transposed into Rhodococcus cells by means of electroporation, either individually in two consecutive processes or in combinations by a single step. During transposition, each of the two vectors liberates its transposable-marker gene, which integrated in a single copy into a random site in the Rhodococcus chromosomal DNA. Southern blot analysis indicated that the two transposable-marker genes of both vectors does not alter or knock out each other. To utilize these vectors for Streptomyces proteasome expression, two expression cassettes were constructed; each cassette comprised a constitutive promoter (P(nit)), the DNA fragment, prcA or prcB that encodes alpha- or beta-subunits of Streptomyces proteasome, and T(thcA) transcriptional terminator. The cassettes were then individually introduced into the multiple cloning sites that are located in the transposable-marker gene of the two vectors. The two cassettes-harboring vectors were subsequently co-transposed, in combinations, into the Rhodococcus genome by a single electroporation step and the Streptomyces proteasome was successfully expressed in the rodococcal host cell. The isolated proteasome was further characterized and the peptidase activity was confirmed and indicated that it was biologically active. The present study concluded that both pTNR-KA and pTNR-TA can be used as transposon-based protein expression systems in Rhodococcus species.

Animals↗

Hepatitis B virus enhances transduction of human hepatocytes by SV40-based vectors.

BACKGROUND/AIMS: Chronic HBV infection, a world-wide epidemic, can lead to chronic hepatitis and eventually to cirrhosis and hepatocellular carcinoma. The liver poses obstacles for many available gene-transfer vectors. SV40-based vectors can transduce human hepatic and hematopoietic cells. We studied the effect of HBV on the transduction - efficiency of human hepatic cells by SV40 - based vectors. METHODS: A SV40-vector carrying the luciferase gene, and wild-type SV40, were used to assess transduction efficiency of human HBV-positive and HBV-negative hepatic cells. Transduction efficiency was measured as luciferase activity or by T-antigen staining. To evaluate whether differences in transduction efficiency are due to cell recognition and/or nuclear transport, MHC-I receptors were measured by FACS analysis and SV40-DNA was extracted from the nuclei of transduced cells and quantified. RESULTS: Two HBV-positive cell-lines, HepG2.2.2.15 and FLC4-A10II, were transduced significantly more efficiently than their parental HBV-negative cell-lines. Transient transfection of HuH-7 cells with the HBV genome also increased transduction efficiency. The level of MHC-I, the cellular receptor for SV40, was comparable in all the cell-lines studied. However, soon after infection with SV40, the nuclei of HepG2.2.2.15 contained >6-fold more SV40-DNA than HepG2. CONCLUSIONS: HBV increases transduction by SV40-vectors. This is due to enhanced vector entry and/or transport into the nucleus. SV40-vectors appear to have a potential for gene therapy for the treatment of HBV infections.

Cell Line↗

Centrifugation enhances integrin-mediated transduction of dendritic cells by conventional and RGD-modified adenoviral vectors.

The level of antigen loading can impact on the capacity for dendritic cells (DC) to activate T cell responses. Several different approaches to adenoviral (Ad)-based transduction were therefore assessed for their effect on both transgene expression and T cell activation. While a conventional E1(-)/E3Delta Ad vector (Ad/GFP) produced a concentration-dependent expression of GFP, a modified vector expressing Arginine-Glycine-Aspartic Acid (RGD) sequence on its fiber knob (Ad-RGD/GFP) enhanced transgene expression by 9-20-fold at each MOI. The addition of centrifugal force (2000xg) during DC transduction with Ad/GFP also increased expression up to 20-fold. However, combining centrifugation with the Ad-RGD/GFP vector produced no effect on transduction rate and only a 1.5- to 2-fold increase in GFP expression, suggesting overlapping mechanisms of action. Consistent with this, exogenous RGD peptide blocked transduction regardless of the vector used, or the addition of centrifugal force, and transduction was primarily limited to DC expressing the CD51 integrin receptor. Ad vectors expressing ovalbumin (OVA) were used to assess transduced DC for their capacity to activate OVA-specific T cells. We observed a significant relationship between transgene expression and the capacity for T cell activation regardless of whether transgene expression was increased by using a higher MOI, an RGD-modified vector, or by employing centrifugal force. Furthermore, combining these approaches produced synergistic effects on T cell activation. We conclude that RGD-modified vectors and centrifugation both enhance DC transduction by increasing entry via integrin receptors and that the capacity for T cell activation can be optimized by combining approaches to achieve the highest possible level of transgene expression.

Adenoviridae↗

Transduction of motor neurons and muscle fibers by intramuscular injection of HIV-1-based vectors pseudotyped with select rabies virus glycoproteins.

For studies of motor neuron function or for therapeutic purposes, novel pseudotype HIV-1-based vectors were developed that are capable of expressing transgenes in motor neurons following injection into mouse hind limb muscles. To specifically target motor neurons, glycoproteins from two rabies virus (RV) isolates, the mouse-brain adapted challenge virus 24 (CVS-24) variants, CVS-N2c and CVS-B2c were evaluated for pseudotype formation with an HIV-1-based vector. Both RV glycoproteins incorporated into vector envelopes, and both pseudotypes yielded high titers with Hek293T and cortical plate neuron cultures. Increased neuronotropism by the CVS-N2c pseudotype was not observed, suggesting that vector tropism is not solely determined by the fusogenic viral glycoprotein. Vector injection into hind limb muscles resulted in EYFP reporter gene expression in the injected muscle fibers and in spinal cord motor neurons innervating the same muscle, indicating retrograde vector transport. Intramuscular vector injections into the soleus and tibialis anterior muscles transduced 26% and 16% of all motor neurons in each motor nucleus, respectively. These transduction efficiencies may allow novel approaches to functional studies of the motor system and the treatment of neuromuscular disease.

Animals↗

Targeted gene transfer to nigrostriatal neurons in the rat brain by helper virus-free HSV-1 vector particles that contain either a chimeric HSV-1 glycoprotein C-GDNF or a gC-BDNF protein.

Direct gene transfer into neurons has potential for both studying neuronal physiology and for developing gene therapy treatments for specific neurological conditions. Due to the heterogeneous cellular composition of the brain, cell-type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. The two prevalent approaches for achieving cell-type-specific expression are to use a cell-type-specific promoter to control recombinant gene expression or to modify a virus vector particle to target gene transfer to a specific cell type. Targeted gene transfer to multiple peripheral cell types has been described, but targeted gene transfer to a specific type of neuron in the brain has yet to be reported. Targeted gene transfer approaches with Herpes Simplex Virus (HSV-1) vectors have focused on modifying glycoprotein C (gC) to remove the heparin binding domain and add a binding activity for a specific protein on the cell surface. This study was designed to develop HSV-1 vectors that target gene transfer to cells that contain receptors for either glial-cell-line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF), such as nigrostriatal neurons. We isolated chimeric gC-GDNF or chimeric gC-BDNF constructs, and the resulting proteins were incorporated into HSV-1 virus particles. We performed helper virus-free HSV-1 vector packaging in the presence of each chimeric protein. The resulting vector stocks supported 2.2- to 5.0-fold targeted gene transfer to nigrostriatal neurons in the rat brain, compared to vector particles that contained wild-type (wt) gC. Gene transfer to nigrostriatal neurons by vector particles that contained chimeric gC-BDNF was reduced by preincubation with an anti-BDNF antibody. Targeted gene transfer to neurons that contain specific neurotrophic factor receptors may benefit specific physiological and gene therapy studies.

Animals↗