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Characterization of the vaccinia MVA hemagglutinin gene locus and its evaluation as an insertion site for foreign genes.

The 'Modified Vaccinia Ankara' (MVA) strain is a potential live vaccine vector. The use of the hemagglutinin (ha) gene of the MVA strain as an insertion site for foreign genes was evaluated. To identify the molecular basis of the hemagglutinin-negative (HA-) phenotype of MVA, the ha gene and the region around this gene were sequenced. Amino acid (aa) sequence comparisons with functional hemagglutinins of other vaccinia strains predicted a functional polypeptide. The late part of the promoter region of the ha gene, however, was deleted, causing the apparent loss of the ha gene function. Nevertheless, insertion of foreign DNA into the ha gene allowed generation of functional recombinant viruses, indicating that the ha-gene region is a suitable insertion site.

Base Sequence↗

Management of herbicide-tolerant oilseed rape in Europe: a case study on minimizing vertical gene flow.

The potential commercialization of genetically modified herbicide-tolerant (GMHT) oilseed rape in Europe raises various concerns about their potential environmental and agronomic impacts, especially those associated with the escape of transgenes. Pollen of oilseed rape can be dispersed in space, resulting in the fertilization of sympatric compatible wild relatives (e.g. Brassica rapa) and oilseed rape cultivars grown nearby (GM and/or non-GM Brassica napus). The spatial and temporal dispersal of seeds of oilseed rape may lead to feral oilseed rape populations outside the cropped areas and oilseed rape volunteers in subsequent crops in the rotation. The incorporation of a HT trait(s) may increase the fitness of the recipient plants, making them more abundant and persistent, and may result in weeds that are difficult to control by the herbicide(s) to which they are tolerant. Vertical gene flow from transgenic oilseed rape to non-GM counterparts may also have an impact on farming and supply chain management, depending on labelling thresholds for the adventitious presence of GM material in non-GM products. Given the extent of pollen and seed dispersal in oilseed rape, it is obvious that the safe and sound integration of GMHT oilseed rape in Europe may require significant on-farm and off-farm management efforts. Crucial practical measures that can reduce vertical gene flow include (1) isolating seed production of Brassica napus, (2) the use of certified seed, (3) isolating fields of GM oilseed rape, (4) harvesting at the correct crop development stage with properly adjusted combine settings, (5) ensuring maximum germination of shed seeds after harvest, (6) controlling volunteers in subsequent crops, and (7) keeping on-farm records. The implementation of the recommended practices may, however, be difficult, entailing various challenges.

Agriculture↗

Capsid modifications overcome low heterogeneous expression of heparan sulfate proteoglycan that limits AAV2-mediated gene transfer and therapeutic efficacy in human ovarian carcinoma.

OBJECTIVES: Capsid-modified AAV vectors can mediate enhanced gene transfer to neoplasms characterized by low AAV receptor expression. Here we sought to determine the therapeutic potential of a capsid-modified AAV vector for gene therapy of ovarian carcinoma (OvCa). METHODS: We tested a panel of OvCa cell lines for AAV2-mediated gene transduction and for sensitivity to ganciclovir (GCV) following AAVHSVtk administration. Levels of AAV internalization and attachment receptor were assessed by flow cytometry and immunohistochemistry. The role of receptors in AAV-mediated gene transfer was assessed by competition assays. Finally, we examined the ability of a modified vector with an integrin-binding RGD motif inserted into the AAV capsid to improve gene delivery to OvCa and enhance AAVHSVtk/GCV-mediated killing by cytotoxicity assay. RESULTS: All OvCa cell lines were poorly transduced with AAV2 vectors and showed variably sensitive to AAVHSVtk/GCV. While OvCa cell lines expressed AAV2 internalization receptors (alphav integrins), expression of the AAV2 attachment receptor, HSPG, was variable and not detected on many lines. Analysis of archived clinical specimens showed no detectable HSPG expression on approximately 45% of primary human tumors. Gene transfer to OvCa was increased several fold using the RGD-modified vector. Gene transfer was independent of HSPG and specific to the targeted receptor. Importantly, the RGD-modified capsid markedly increased the ability of the AAVHSVtk to kill OvCa cells in the presence of GCV. CONCLUSIONS: The development of AAV vectors targeted to cell surface receptors other than HSPG will be critical to the advancement of AAV-mediated gene therapy for treating OvCa.

Adenoviridae↗

Effects of recombinant adenovirus-mediated expression of IL-2 and IL-12 in human B lymphoma cells on co-cultured PBMC.

BACKGROUND: Modulation of the immune system by genetically modified lymphoma cell vaccines is of potential therapeutic value in the treatment of B cell lymphoma. However, the anti-tumor effect of any single immunogene transfer has so far been limited. Combination treatment of recombinant IL-2 and IL-12 has been reported to be synergistic for inducing anti-tumor responses in solid tumors but the potential of IL-2/IL-12 gene modified B cell lymphoma cells has not been explored yet. METHODS: Using three different human B cell lymphoma cell lines and primary samples from patients with B cell neoplasms, expression levels of the coxsackie B-adenovirus receptor (CAR) and alpha (v) integrins were analyzed by fluorescence-activated cell sorter (FACS). Adenoviral transduction efficiencies were determined by GFP expression analysis and IL-2 and IL-12 cytokine production was quantified by enzyme-linked immunosorbent (ELISA) assays. Proliferative activities of peripheral blood mononuclear cells (PBMC) stimulated with either cytokine derived from supernatants of transduced lymphoma cells were measured by cell proliferation (MTT) assays. An EuTDA cytotoxicity assay was used to compare cytotoxic activities of IL-2 and/or IL-12 stimulated PBMC against unmodified lymphoma cells. RESULTS: We found that B cell lymphoma cell lines could be transduced with much higher efficiency than primary tumor samples, which appeared to correlate with the expression of CAR. Adenoviral-expressed IL-2 and IL-12 similarly led to dose-dependent increases in proliferation rates of PBMC obtained from healthy donors. IL-2 and/or IL-12 transduced lymphoma cells were co-cultured with PBMC, which were assayed for their cytolytic activity against unmodified lymphoma cells. We found that IL-2 stimulated PBMC elicited a significant anti-tumor effect but not the combined effect of IL-2/IL-12 or IL-12 alone. CONCLUSION: This study demonstrates that the generation of recombinant adenovirus modified lymphoma cell vaccines based on lymphoma cell lines expressing IL-2 and IL-12 cytokine genes is technically feasible, induces increases in proliferation rates and cytotoxic activity of co-cultured PBMC, and warrants further development for the treatment of lymphoma patients in the future.

Journal Article↗

Retroviral transduction of hematopoietic progenitor cells with mutant p53 promotes survival and proliferation, modifies differentiation potential and inhibits apoptosis.

Mutations in the p53 tumor suppressor gene have been shown to be associated with many human tumors and various leukemias and lymphomas. To examine whether constitutive overexpression of mutant p53 can effect transformation of normal hematopoietic cells, a mutant p53 gene was introduced into normal murine bone marrow hematopoietic cells by retroviral gene transfer. Compared to vector alone-infected cells, hematopoietic progenitor cells transduced with mutant p53 showed increased proliferative potential, enhanced cloning efficiencies and a modified differentiation pattern in vitro. In addition, mutant p53-transduced hematopoietic cells were more resistant to loss of viability and/or induction of apoptosis when cultured in a low concentration of serum or in the absence of both growth factors and serum. These effects occurred rapidly with no apparent contributory secondary events. No permanent cell lines or growth factor-independent cell strains were obtained. The results indicate that introduction of mutant p53 into normal hematopoietic cells in vitro contributes to transformation, including enhanced proliferative potential, modified differentiation and the suppression of apoptosis in these cells.

Animals↗

Variable phenotypic presentation of iron overload in H63D homozygotes: are genetic modifiers the cause?

BACKGROUND: First considered as a polymorphism of the HFE gene, the H63D mutation is now widely recognised as a haemochromatosis associated allele. But few H63D homozygotes with clinical manifestations of hereditary haemochromatosis (HH) have been reported. Concurrently, an increasing number of genes have been shown to interact with HFE in iron metabolism. AIMS: To describe the clinical expression of iron overload (IO) associated with H63D homozygosity, and search for potential genetic modifiers (within the HFE or other genes) that could explain the variability of the phenotypes. PATIENTS AND METHODS: We retrospectively analysed the clinical phenotype of 56 H63D homozygotes referred for a personal or family history of IO. For each subject we examined intragenic HFE haplotypes and transferrin receptor (TfR) gene polymorphisms and searched for the Y250X mutation on the TFR2 gene. Additionally, we sequenced the HFE gene of H63D homozygotes with HH. RESULTS: Fifty of 56 subjects had biological and/or clinical abnormalities of iron metabolism. Up to two thirds of patients (n=34) had no acquired cause of IO. Among these, 12 had a phenotypic diagnosis of HH. In the iron loaded group there was a strong prevalence of male patients. No correlation was found between the potential genetic modifiers and phenotypes. No additional mutation of HFE was identified. CONCLUSION: The variable phenotypes associated with H63D homozygosity do not appear to be linked to other HFE mutations, to the TFR2 Y250X mutation, or to HFE or TfR gene intragenic polymorphisms. The exact role of H63D homozygosity in IO and HH needs to be further investigated in unselected populations.

Adult↗

Effects of A1 adenosine receptor overexpression on normoxic and post-ischemic gene expression.

OBJECTIVES: To identify potential molecular genetic determinants of cardiovascular ischemic tolerance in wild-type and transgenic hearts overexpressing A(1) adenosine receptors (A(1)ARs). METHODS: cDNA microarrays were used to explore expression of 1824 genes in wild-type hearts and ischemia-tolerant mouse hearts overexpressing A(1)ARs. RESULTS: Overexpression of A(1)ARs reduced post-ischemic contractile dysfunction, limited arrhythmogenesis, and reduced necrosis by approximately 80% in hearts subjected to 30 min global ischemia 60 min reperfusion. Cardioprotection was abrogated by acute A(1)AR antagonism, and only a small number (19) of genes were modified by A(1)AR overexpression in normoxic hearts. Ischemia-reperfusion significantly altered expression of 75 genes in wild-type hearts (14 induced, 61 down-regulated), including genes for metabolic enzymes, structural/motility proteins, cell signaling proteins, defense/growth proteins, and regulators of transcription and translation. A(1)AR overexpression reversed the majority of gene down-regulation whereas gene induction was generally unaltered. Additionally, genes involved in cell defence, signaling and gene expression were selectively modified by ischemia in transgenic hearts (33 induced, 10 down-regulated), possibly contributing to the protected phenotype. Real-time PCR verified changes in nine selected genes, revealing concordance with array data. Transcription of the A(1)AR gene was also modestly reduced post-ischemia, consistent with impaired functional sensitivity to A(1)AR stimulation CONCLUSIONS: Data are presented regarding the early post-ischemic gene profile of intact heart. Reduced A(1)AR transcription is observed which may contribute to poor outcome from ischemia. A(1)AR overexpression selectively modifies post-ischemic gene expression, potentially contributing to ischemic-tolerance.

Animals↗

A retroviral vector containing a muscle-specific enhancer drives gene expression only in differentiated muscle fibers.

Genetically modified myogenic cells have a number of potentially relevant applications for gene therapy of genetic defects. Retroviral vectors proved to be a safe and efficient tool to transfer and express genes into satellite cells and their differentiated progeny, although muscle-specific regulation of the transferred gene is very difficult to achieve in a conventional vector framework. We modified a Moloney murine leukemia virus (MoMLV)-derived retroviral vector containing a bacterial beta-galactosidase (beta-Gal) reporter gene by inserting a muscle creatinine kinase (MCK) enhancer element into the U3 region of the viral long terminal repeat (LTR). The resulting vector (mLBSN) was transferred into cells of different histological origin, including undifferentiated murine and human myogenic cells, which were unable to express the transgene at detectable levels. Instead, gene expression from the modified LTR was obtained in a mouse myogenic cell line and in human primary satellite cells upon induction of differentiation into myotubes in culture, and correlated with the activation of the muscle differentiation program. beta-Gal-negative, mLBSN-transduced human satellite cells were also transplanted into the quadricep muscle of immunodeficient mice, where activation of the transgene expression was observed in vivo after differentiation and fusion into muscle fibers. These results show that retroviral vectors carrying LTRs modified in the enhancer sequences may be used to target tissue- and differentiation-specific gene expression into the muscle. For practical purposes, satellite cells engineered by muscle-specific retroviral vectors might represent an effective tool to deliver expression of a given gene product specifically into the muscle tissue, avoiding undesired protein accumulation in mononucleated cells. More generally, this type of vector might be useful whenever regulated expression of a transferred gene is necessary in a target cell or tissue.

Animals↗

Conservation biology, genetically modified organisms, and the biosafety protocol.

Concerns have been raised regarding the potential adverse effects on biological diversity of the use of living modified organisms (LMOs, which are commonly known by similar terms such as genetically modified organisms). At the international level these concerns are addressed in part by an agreement known as the Cartagena Protocol on Biosafety and include potential toxic effects of insect-resistant crops on nontarget organisms and potential ecological effects of gene flow from modified crops, fish, microorganisms, or insects to wild species or counterparts. We reviewed the protocol's main provisions, including those dealing with risk assessment and risk management, decision making on imports, documentation accompanying shipments, and liability resulting from damages caused by LMOs. A medium-term program of work has been adopted by the parties, which includes the potential contribution of conservation biologists to delivering capacity building, developing risk assessment guidance, evaluating mechanisms of potential ecological damages from LMOs, and other issues. Conservation biologists and other experts have opportunities to influence the negotiations and implementation of the protocol by providing inputs at meetings, offering expertise to governments and organizations, and participating in or developing relevant projects and initiatives. Involvement of conservation biologists in the implementation and further development of the protocol would contribute to its effectiveness.

Animals↗

Gene therapy of B-cell lymphoma with cytokine gene-modified trioma cells.

The trioma approach is a new immunotherapeutic strategy for treating B-cell lymphomas. It is based on converting the tumour idiotype to a bispecific immunoglobulin that redirects the idiotype to antigen-presenting cells. We show here that even pre-existing tumours can be eradicated by trioma vaccination, that the trioma approach is superior to vaccination with cytokine gene-modified autologous tumour cells and that there is a synergism between trioma immunisation and GM-CSF gene transfer. Furthermore, we show that the immunising potential of GM-CSF gene-modified autologous lymphoma cells is not as dependent on the cytokine expression level as described for other tumour models, such that even minute expression rates are effective. IL-4 gene transfer in the lymphoma model is considerably less efficient or even ineffective when more sensitive systems are used. Remarkably, trioma-mediated effects are extinguished when IL-4 is expressed by the trioma cell.

Animals↗

Hematopoietic stem cell gene therapy: progress toward therapeutic targets.

The concept of hematopoietic stem cell gene therapy is as exciting as that of stem cell transplantation itself. The past 20 years of research have led to improved techniques for transferring and expressing genes in hematopoietic stem cells and preclinical models now routinely indicate the ease with which new genes can be expressed in repopulating stem cells of multiple species. Both modified murine oncoretroviruses and lentiviruses transmit genes into the genome of hematopoietic stem cells and allow expression in the host following transplantation. Using oncoretroviruses, therapeutic genes for severe combined immunodeficiency, common variable gamma chain immunodeficiency, chronic granulomatous disease, Hurler's and Gaucher's Disease have all been used clinically with only modest success except for the patients with immunodeficiency in whom a partial T-cell chimerism has been dramatic. Since stem cell selection in vivo appears important to the therapeutic success of gene transfer, drug resistance selection, most recently using the MGMT gene, has been developed and appears to be safe. Future trials combining a drug resistance and therapeutic gene are planned, as are trials using safety-modified lentiviruses. The therapeutic potential of hematopoietic stem cell gene therapy, particularly given recent advances in stem cell plasticity, remains an exceptionally exciting area of clinical research.

Genetic Therapy↗

MIM, a potential metastasis suppressor gene in bladder cancer.

Using a modified version of the mRNA differential display technique, five human bladder cancer cell lines from low grade to metastatic were analyzed to identify differences in gene expression. A 316-bp cDNA (C11-300) was isolated that was not expressed in the metastatic cell line TccSuP. Sequence analysis revealed that this gene was identical to KIAA 0429, has a 5.3-kb transcript that mapped to 8q24.1. The protein is predicted to be 356 amino acids in size and has an actin-binding WH2 domain. Northern blot revealed expression in multiple normal tissues, but none in a metastatic breast cancer cell line (SKBR3) or in metastatic prostatic cancer cell lines (LNCaP, PC3). We have named this gene Missing in Metastasis (MIM) and our data suggest that it may be involved in cytoskeletal organization.

Amino Acid Sequence↗

Bactrocera tryoni and closely related pest tephritids--molecular analysis and prospects for transgenic control strategies.

Bactrocera tryoni is a serious pest of horticulture in eastern Australia. Here we review molecular data relevant to pest status and development of a transformation system for this species. The development of transformation vectors for non-drosophilid insects has opened the door to the possibility of improving the sterile insect technique (SIT), by genetically engineering factory strains of pest insects to produce male-only broods. Transposition assays indicate that all five of the vectors currently used for transformation in non-drosophilid species have the potential to be useful as transformation vectors in B. tryoni. Evidence of cross mobilization of hobo by an endogenous Homer element emphasises the necessity to understand the endogenous transposons within a species. The sex-specific doublesex and yolk protein genes have been characterized with a view to engineering a female-specific lethal gene or modifying gene expression through RNA interference (RNAi). Data are presented which indicate the potential of RNAi to modify the sex ratio of resultant broods. An understanding of how pest status is determined and maintained is being addressed through the characterization of genes of the circadian clock that enable the fly to adapt to environmental cues. Such an understanding will be useful in the future to the effective delivery of sophisticated pest control measures.

Animals↗

Natural killer cell-mediated lysis of autologous cells modified by gene therapy.

This study investigated the role of natural killer (NK) cells as effectors of an immune response against autologous cells modified by gene therapy. T lymphocytes were transduced with LXSN, a retroviral vector adopted for human gene therapy that carries the selectable marker gene neo, and the autologous NK response was evaluated. We found that (i) infection with LXSN makes cells susceptible to autologous NK cell-mediated lysis; (ii) expression of the neo gene is responsible for conferring susceptibility to lysis; (iii) lysis of neo-expressing cells is clonally distributed and mediated only by NK clones that exhibit human histocompatibility leukocyte antigen (HLA)-Bw4 specificity and bear KIR3DL1, a Bw4-specific NK inhibitory receptor; and (iv) the targets are cells from HLA-Bw4(+) individuals. Finally, neo peptides anchoring to the Bw4 allele HLA-B27 interfered with KIR3DL1-mediated recognition of HLA-B27, i.e., they triggered NK lysis. Moreover, neo gene mutations preventing translation of two of the four potentially nonprotective peptides reduced KIR3DL1(+) NK clone-mediated autologous lysis. Thus, individuals expressing Bw4 alleles possess an NK repertoire with the potential to eliminate autologous cells modified by gene therapy. By demonstrating that NK cells can selectively detect the expression of heterologous genes, these observations provide a general model of the NK cell-mediated control of viral infections.

Amino Acid Sequence↗

Platelet glycoprotein I(b)alpha and integrin alpha2 beta1 polymorphisms: gene frequencies and linkage disequilibrium in a population diversity panel.

Genetic variants in the GP1BA and ITGA2 genes have been proposed as potential modifiers for arterial vascular disease and bleeding disorders. Since ancestry may play an important role in the prevalence of these variants, we sought to determine their allele frequency and linkage disequilibrium in a collection of 1064 DNA samples from 51 ethnic groups. We studied haplotypes of ITGA2 defined by single nucleotide substitutions at positions -52, 807, and 1648, and GP1BA variants defined by sequence changes in positions -5 (Kozak), 1018 (T145M, HPA-2) and 1285 (VNTR A, B, C and D). Frequency of haplotypes of ITGA2 showed considerable variation across the different groups, with a higher prevalence of the haplotype -52C or T/807C/1648A observed in African compared with caucasian and Asian populations. The haplotypes 52C/807T/1648A and -52T/807T/1648A were not observed in caucasians or South Americans. While relative frequencies of the GP1BA Kozak alleles were comparable across groups, the methionine allele (HPA-2b) showed a higher frequency in Africa (0.26) than in the other groups. We also observed a high prevalence of the VNTR B allele in the African and Israeli populations. Haplotype analysis revealed incomplete linkage disequilibrium between the HPA-2 and VNTR alleles. Incorporation of GP1BA variants into the set of SNPs already genotyped by the HapMap project disrupted the pre-existing haplotype block. These data provide a valuable resource for optimal selection of variants best tailored for association studies of vascular disease or bleeding disorders when examining individuals of different ancestral origins.

Alleles↗

Cationic poly(lactide-co-glycolide) nanoparticles as efficient in vivo gene transfection agents.

Poly(lactide-co-glycolide) (PLGA), a biocompatible and biodegradable polyester co-polymer of PLA and PGA, has been recognized for its ability to deliver genes. However, gene delivery by PLGA nanoparticles is limited by their negative charge and their poor transport through mucosal barriers. In this study, PLGA nanoparticles were surface modified with cationic chitosan in an effort to improve their gene delivery capability. PLGA nanoparticles were synthesized by emulsion-diffusion-evaporation technique using PVA-chitosan (PLGA1) or PVA-chitosan-PEG (PLGA2) blend as stabilizers. This method is reproducible and produces nanoparticles with hydrodynamic diameter <200 nm. The nanoparticles were characterized by zetasizer, photon correlation spectroscopy and atomic force microscopy. A549 epithelial cells were transfected in vitro with PLGA particles complexed with a reporter plasmid encoding green fluorescent protein. PLGA particles transferred EGFP gene, but were less efficient than the lipofectamine control. The nanoparticles were also tested for their ability to transport across the nasal mucosa in vivo in mice. The results show that both PLGA1 and PLGA2 facilitate gene delivery and expression in vivo with increased efficiency and without causing inflammation, as measured by IL-6. Together, these results indicate that chitosan-modified PLGA nanoparticles have greater potential as gene carriers.

Animals↗

Glutathione S-transferase T1 null-genotype is associated with an increased risk of lung cancer.

Glutathione S-transferases (GSTs) are involved in detoxification of carcinogens, e.g., from tobacco smoke. Therefore, polymorphisms in the GST genes have been considered as potential modifiers of individual cancer risk. In a population-based case-cohort study where cases and the subcohort sample were matched on duration of smoking, we investigated the occurrence of lung cancer and histological subtypes of lung cancer in relation to deletion polymorphism in both GSTM1 and GSTT1, single nucleotide polymorphisms (SNPs) in GSTP1 (Ile105Val and Ala114Val) and a 3 base pair deletion polymorphism in GSTM3. We further investigated the effects of the GST polymorphisms on lung cancer risk within subgroups of subjects defined by gender and age. The results showed a 2.4-fold (CI = 1.31-4.41) increased risk of lung cancer in GSTT1 null-genotype carriers but no significant effects of the polymorphisms in GSTM1, GSTM3, GSTP1-105 or GSTP1-114. The association was strongest in lower age groups, with a 9.6-fold increase in risk for subjects with the GSTT1 null-genotype in the 50-55 years age interval (CI = 3.03-30.59). Positive associations were found for GSTT1 within all major histological subtypes. Squamous cell carcinoma was the histological type most strongly associated with the GSTT1 genotype, with a 5.0-fold (CI = 2.26-11.18) increase in risk for subjects carrying the GSTT1 null-genotype. The effects of the GSTT1 null-genotype seemed stronger in the presence of the GSTM1 null-genotype or the GSTP1-105 variant allele. These results suggest that the GSTT1 null-genotype is associated with an increased risk of lung cancer, especially in younger individuals.

Aged↗

Versatile peptide dendrimers for nucleic acid delivery.

Dendrimers are nonviral vectors that have attracted interest on account of a number of features. They are structurally versatile because their size, shape, and surface charge can be selectively altered. Here we examine the functions of a new family of composite dendrimers that were synthesized with lipidic amino acid cores. These dendrimers are bifunctional because they are characterized by positively charged (lysine) modules for interaction with nucleic acids and neutral lipidic moieties for membrane lipid-bilayer transit. We assessed their structure-function correlations by a combination of molecular and biophysical techniques. Our assessment revealed an unexpected pleitropy of functions subserved by these vectors that included plasmid and oligonucleotide delivery. We also generated a firefly luciferase cell line in which we could modulate luciferase activity by RNA interference. We found that these vectors could also mediate RNA suppression of luciferase expression by delivering double-stranded luciferase transcripts generated in vitro. The structural uniqueness of these lipidic peptide dendrimers coupled with their ease and specificity of assembly and the versatility in their choice of cargo, puts them in a new category of macromolecule carriers. These vectors, therefore, have potential applications as epigenetic modifiers of gene function.

Animals↗