Transgenic Breeding Research Articles

Differential gene expression during somatic embryogenesis in the maize (Zea mays L.) inbred line H99

Somatic embryogenesis is a complex developmental process that offers great potential for plant propagation. Although many studies have shown that the generation of embryonic cells from somatic cells is accompanied by the synthesis of RNA and DNA and by elevated enzymatic activity, the mechanism of the onset of somatic embryogenesis is not well understood. cDNA-amplified fragment length polymorphism analysis was used to evaluate the gene expression pattern in embryogenic and non-embryogenic of the inbred maize line H99 during the process of embryogenesis. We identified a total of 101 candidate genes associated with the formation of maize embryonic calli. Based on the sequence analysis, these genes included 53 functionally-annotated TDFs involved in such processes as energy production and conversion, cell division and signal transduction, suggesting that somatic embryogenesis undergoes a complex process. Two full-length cDNA sequences, encoding KHCP (kinesin heavy chain like protein) and TypA (tyrosine phosphorylation protein A), and partial sequences, encoding ARF-GEP (guanine nucleotide-exchange protein of ADP ribosylation factor) homologs, were isolated from embryonic calli of maize and named ZmKHCP, ZmTypA and ZmARF-GEP, respectively. Finally, the real-time qRT-PCR results showed that the expression levels of the three genes were significantly higher in the embryonic calli than the non-embryonic calli. Thus, this study provides important clues to understanding the induction of somatic embryogenesis in maize. The candidate genes associated with the formation of embryonic calli may offer additional insights into the mechanism of somatic embryogenesis, and further research on the three candidate genes may determine their role in increasing the rate of induction of embryonic calli, which may aid in the development of cultivars through transgenic breeding.

Establishment of a spleen cell line from large yellow croaker Pseudosciaena crocea and its primitive application in foreign gene transfection

A large yellow croaker, Pseudosciaena crocea, spleen (LYCS) cell line was established and the feasibility of using it for foreign gene transfection was evaluaed in this study. Primary culture of LYCS cells was initiated from spleen tissue pieces, which were cultured at 25°C in Dulbecco’s modiced Eagle medium/F12 medium (DMEM/F12, 1:1) (pH7.2), supplemented with 20% fetal bovine serum, carboxymethyl chitosan, chondroitin sulfate, basic fibroblast growth factor (bFGF) and insulin-like growth factor-I (IGF-I). The cultured LYCS cells, in fibroblast shape, proliferated to 100% confluency 20 days later. Chromosome analyses indicated that the LYCS cells exhibited chromosomal aneuploidy with a modal chromosome number of 48 which displayed the normal diploid karyotype of P. crocea (6m+6sm+36t, NF=60). A LYCS cell line, with a population doubling time of 48.7 h at passage 60, has been established and subcultured to passage 70. Transgenic feasibility test demonstrated that positive green fluorescence protein (GFP) expression was observed in LYCS cells after pcDNA3.1-GFP plasmid transfection. In conclusion, a continuous foreign gene transfection feasible LYCS cell line has been established successfully. The cell line might serve as a valuable tool for studies of transgenic breeding and has potential applications for different kinds of cytotechnological studies.

Zebrafish mll Gene Is Essential for Hematopoiesis

Studies implicate an important role for the mixed lineage leukemia (Mll) gene in hematopoiesis, mainly through maintaining Hox gene expression. However, the mechanisms underlying Mll-mediated hematopoiesis during embryogenesis remain largely unclear. Here, we investigate the role of mll during zebrafish embryogenesis, particularly hematopoiesis. Mll depletion caused severe defects in hematopoiesis as indicated by a lack of blood flow and mature blood cells as well as a significant reduction in expression of hematopoietic progenitor and mature blood cell markers. Furthermore, mll depletion prevented the differentiation of hematopoietic progenitors. In addition, we identified the N-terminal mini-peptide of Mll that acted as a dominant negative form to disrupt normal function of mll during embryogenesis. As expected, mll knockdown altered the expression of a subset of Hox genes. However, overexpression of these down-regulated Hox genes only partially rescued the blood deficiency, suggesting that mll may target additional genes to regulate hematopoiesis. In the mll morphants, microarray analysis revealed a dramatic up-regulation of gadd45αa. Multiple assays indicate that mll inhibited gadd45αa expression and that overexpression of gadd45αa mRNA led to a phenotype similar to the one seen in the mll morphants. Taken together, these findings demonstrate that zebrafish mll plays essential roles in hematopoiesis and that gadd45αa may serve as a potential downstream target for mediating the function of mll in hematopoiesis.

Application of a high-speed breeding technology to apple (Malus × domestica) based on transgenic early flowering plants and marker-assisted selection

Breeding of apple (Malus × domestica) remains a slow process because of protracted generation cycles. Shortening the juvenile phase to achieve the introgression of traits from wild species into prebreeding material within a reasonable time frame is a great challenge. In this study, we evaluated early flowering transgenic apple lines overexpressing the BpMADS4 gene of silver birch with regard to tree morphology in glasshouse conditions. Based on the results obtained, line T1190 was selected for further analysis and application to fast breeding. The DNA sequences flanking the T-DNA were isolated and the T-DNA integration site was mapped on linkage group 4. The inheritance and correctness of the T-DNA integration were confirmed after meiosis. A crossbred breeding programme was initiated by crossing T1190 with the fire blight-resistant wild species Malus fusca. Transgenic early flowering F(1) seedlings were selected and backcrossed with 'Regia' and 98/6-10 in order to introgress the apple scab Rvi2, Rvi4 and powdery mildew Pl-1, Pl-2 resistance genes and the fire blight resistance quantitative trait locus FB-F7 present in 'Regia'. Three transgenic BC'1 seedlings pyramiding Rvi2, Rvi4 and FB-F7, as well as three other BC'1 seedlings combining Pl-1 and Pl-2, were identified. Thus, the first transgenic early flowering-based apple breeding programme combined with marker-assisted selection was established.

Cloning and characterization of a maize SnRK2 protein kinase gene confers enhanced salt tolerance in transgenic Arabidopsis

SnRK2 (sucrose non-fermenting 1-related protein kinases 2) represents a unique family of protein kinase in regulating signaling transduction in plants. Although the regulatory mechanisms of SnRK2 have been well demonstrated in Arabidopsis thaliana, their functions in maize are still unknown. In our study, we cloned an SnRK2 gene from maize, ZmSAPK8, which encoded a putative homolog of the rice SAPK8 protein. ZmSAPK8 had two copies in the maize genome and harbored eight introns in its coding region. We demonstrated that ZmSAPK8 expressed differentially in various organs of maize plants and was up-regulated by high-salinity and drought treatment. A green fluorescent protein (GFP)-tagged ZmSAPK8 showed subcellular localization in the cell membrane, cytoplasm and nucleus. In vitro kinase assays indicated that ZmSAPK8 preferred Mn(2+) to Mg(2+) as cofactor for phosphorylation, and Ser-182 and Thr-183 in activation loop was important for its activity. Heterologous overexpression of ZmSAPK8 in Arabidopsis could significantly strengthen tolerance to salt stress. Under salt treatment, ZmSAPK8-overexpressed transgenic plants exhibited higher germination rate and proline content, low electrolyte leakage and higher survival rate than wild type. Further analysis indicated that transgenic plants showed increased transcription of the stress-related genes, RD29A, RD29B, RAB18, ABI1, DREB2A and P5CS1, under high-salinity conditions. The results demonstrated that ZmSAPK8 was involved in diverse stress signal transduction. Moreover, no obvious adverse effects on growth and development in the ZmSAPK8-overexpressed transgenic plants implied that ZmSAPK8 was potentially useful in transgenic breeding to improve salt tolerance in crops.

Indication of the Expression of Transgene in Rice Plant Based on Hyperspectral Remote Sensing Technique II——Growth Monitoring of Samples in the Contrast Experiment

Since the complication of monitoring and evaluating the problems about the transgenic expression and its impacts on the receptor in the transgenic crop breeding and other relevant evaluated works, the authors in the present work tried to assess the differences of spectral parameters of the transgenic rice in contrast with its parent group quantitatively and qualitatively, fulfilling the growth monitoring of the transgenic samples. The spectral parameters (spectral morphological characteristics and indices) chosen are highly related to internal or external stresses to the receipts, and thus could be applied as indicators of biophysical or biochemical processes changes of plant. By ASD portable field spectroradiometer with high-density probe, fine foliar spectra of 8 groups were obtained. By analyzing spectral angle and continuum removal, the spectral morphological differences and their locations of sample spectra were found which could be as auxiliary priori knowledge for quantitative analysis. By investigating spectral indices of the samples, the quantitative differences of spectra were revealed about foliar chlorophyll a+b and carotenoid content. In this study both the spectral differences between transgenic and parent groups and among transgenic groups were investigated. The results show that hyperspectral technique is promising and a helpful auxiliary tool in the study of monitoring the transgenic crop and other relevant researches. By this technique, quantitative and qualitative results of sample spectra could be provided as prior knowledge, as certain orientation, for laboratory professional advanced transgenic breeding study.

Advances of transgenic breeding in livestock

Transgenic technology represents a revolutionary way to produce elite livestock breeds, allowing introduction of alien gene into livestock genome. Currently, pronuclear microinjection of DNA and somatic cell nuclear transfer are two popular methods used to make transgenic farm animals. Transgenic technology can be used in livestock breeding for improving disease resistance, carcass composition, lactational performance, wool production, growth rate, and reproductive performance, as well as reducing negative environmental impact. In addition to introduction of animal transgenic technologies, this review described the status and the future perspective of transgenic breeding in livestock.

Inheritance and expression stability of transgene in transgenic animals

Transgenic technology is one of the most hotspots in biology. In the past decade, the progress in animal cloning has provided an alternative method to improve transgenic efficiency. Many kinds of transgenic animals have been successfully produced via the combination of transfection and nuclear transfer. However, the ultimate aim of transgenesis is not to produce several transgenic animals, but to service for the needs of human. In animal production, transgenic technology has been used to breed new livestock, which has received a lot of attention in China. It has been evidenced that inheritance and expression instability of transgene in transgenic animals is still the major limitation, which is attributed to position effect, epigenetic modification, and hereditary efficiency of transgene. In this review, we discussed the three points for promoting the industrialization of animal transgenic breeding.

Transgenic expression of TaMYB2A confers enhanced tolerance to multiple abiotic stresses in Arabidopsis

Osmotic stresses such as drought, salinity, and cold are major environmental factors that limit agricultural productivity. Transcription factors play essential roles in abiotic stress signaling in plants. Three TaMYB2 members were identified and designated TaMYB2A, TaMYB2B, and TaMYB2D based on their genomic origins. The cis-regulatory elements in the promoter regions were compared, and their diverse expression patterns under different abiotic stress conditions were identified. TaMYB2A was further characterized because of its earlier response to stresses. Subcellular localization revealed that TaMYB2A localized in the nucleus. To examine the role of TaMYB2A under various environmental stresses, transgenic Arabidopsis plants carrying TaMYB2A controlled by the CaMV 35S promoter were generated and subjected to severe abiotic stress. TaMYB2A transgenics had enhanced tolerance to drought, salt, and freezing stresses, which were confirmed by the enhanced expressions of abiotic stress-responsive genes and several physiological indices, including decreased rate of water loss, enhanced cell membrane stability, improved photosynthetic potential, and reduced osmotic potential. TaMYB2A is a multifunctional regulatory factor. Its overexpression confers enhanced tolerance to multiple abiotic stresses while having no obvious negative effects on phenotype under well-watered and stressed conditions; thus, TaMYB2A has the potential for utilization in transgenic breeding to improve abiotic stress tolerances in crops.

PdERECTA, a leucine-rich repeat receptor-like kinase of poplar, confers enhanced water use efficiency in Arabidopsis

Water deficiency causes a dramatic reduction in crop production globally. Breeding crop varieties that are more efficient in their water use is one strategy to overcome this predicament. In this study, a member of the LRR-RLKs family, the Populus nigra×(Populus deltoides×Populus nigra) ERECTA (PdERECTA) gene was cloned. To study the biological functions of PdERECTA, transgenic Arabidopsis plants (35S:PdERECTA) that constitutively expressed the PdERECTA gene were constructed. Overexpression of PdERECTA resulted in early seedling establishment, longer primary roots, and larger leaf areas. Notably, transgenic Arabidopsis overexpressing PdERECTA resulted in enhanced long-term water use efficiency (WUEl), as estimated by the analysis of carbon isotopic discrimination. The WUEl results were supported by the physiological and anatomical results, which included improved photosynthetic rate, decreased transpiration rate, and stomatal density. The transgenic lines have significantly more dry-biomass as compared to the wild type. Since the overexpression of PdERECTA can strongly enhance the water use efficiency in transgenic Arabidopsis plants, PdERECTA could potentially be used in transgenic breeding to improve the water use efficiency.

Overexpression of transcription factor ZmPTF1 improves low phosphate tolerance of maize by regulating carbon metabolism and root growth

A bHLH (basic helix-loop-helix domain) transcription factor involved in tolerance to Pi starvation was cloned from Zea mays with an RT-PCR coupled RACE approach and named ZmPTF1. ZmPTF1 encoded a putative protein of 481 amino acids that had identity with OsPTF1 in basic region. Real-time RT-PCR revealed that ZmPTF1 was quickly and significantly up-regulated in the root under phosphate starvation conditions. Overexpression of ZmPTF1 in maize improved root development, enhanced biomass both in hydroponic cultures and sand pots, and the plants developed more tassel branches and larger kernels when they were grown in low phosphate soil. Compared with wild type, overexpressing ZmPTF1 altered the concentrations of soluble sugars in transgenic plants, in which soluble sugars levels were lower in the leaves and higher in the roots. Overexpression of ZmPTF1 enhanced the expression of fructose-1,6-bisphosphatase and sucrose phosphate synthase1 participated in sucrose synthesis in the leaves but decreased them in the root, and reduced the expression of genes involved in sucrose catabolism in the roots. The modifications on the physiology and root morphology of the plants enhanced low phosphate tolerance and increased the yield under low phosphate conditions. This research provides a useful gene for transgenic breeding of maize that is tolerant to phosphate deficiency and is helpful for exploring the relationship between sugar signaling and phosphate concentrations in cells.

Gene Expression and Functional Genomic Approach for abiotic stress tolerance in different crop species

Abiotic stresses like drought, cold, heat and high salinity are the most damaging factors to agricultural productivity worldwide. Functional genomics has played a key role in making plant breeding more efficient in development of crop varieties tolerant to biotic and abiotic stresses. Numerous genes and their products respond to these stresses at transcriptional and translational level. Although many drought responsive genes have been discovered, it is still of great importance to analyse drought-inducible genes and their expression in drought-tolerant crops. In addition to various molecular markers, Expressed Sequenced Tags (ESTs) are currently used as a fast and efficient method of profiling genes expressed in various tissues, cell types or developmental stages. The genes, thus, discovered will be utilized in their transfer to commercially important crops through marker assisted selection or transgenic breeding. This review discusses the techniques currently most in use in gene expression analysis, together with some important experimental design variables, such as the developmental stage of the plant, stress intensity and duration, and how different stresses may interact when performing assays.

Construction of Plant Expression Constructs Harboring Full-length <i>Bt</i> Cry1Ac22 Toxin Gene and Truncated Functional Domains of <i>Bt</i> Cry1Ac22 Toxin and Arabidopsis Transformation

The full-length Bt toxin gene (3 534 bp) and truncated functional domains of Bt toxin gene (1 959 bp) of cry1Ac22 were amplified by PCR from Bt strain W015-1. The PCR products were ligated into plant expression vector pBI121 cutting off the s-glucuronidase gene to make the constructs of pBI121-Cry1Ac22F and pBI121-Cry1Ac22T. The constructs carrying with Kanamycin resistant marker were transferred into T-DNA vector and then validated by restricted enzyme digestion and PCR identification. Arabidopsis transformation with the transfered T-DNA vector were performed during the flowering stage mediated by Agrobacterium tumufaciens . Transgenic Arabidopsis seeds with positive Kanamycin resistance were harvested in this research that facilitate the understanding of Bt toxin functions in plant transgenic breeding.

Characterization of a common wheat (Triticum aestivum L.) TaSnRK2.7 gene involved in abiotic stress responses

Sucrose non-fermenting-1-related protein kinase 2 (SnRK2) plays a key role in the plant stress signalling transduction pathway via phosphorylation. Here, a SnRK2 member of common wheat, TaSnRK2.7, was cloned and characterized. Southern blot analysis suggested that the common wheat genome contains three copies of TaSnRK2.7. Subcellular localization showed the presence of TaSnRK2.7 in the cell membrane, cytoplasm, and nucleus. Expression patterns revealed that TaSnRK2.7 is expressed strongly in roots, and responds to polyethylene glycol, NaCl, and cold stress, but not to abscisic acid (ABA) application, suggesting that TaSnRK2.7 might participate in non-ABA-dependent signal transduction pathways. TaSnRK2.7 was transferred to Arabidopsis under the control of the CaMV-35S promoter. Function analysis showed that TaSnRK2.7 is involved in carbohydrate metabolism, decreasing osmotic potential, enhancing photosystem II activity, and promoting root growth. Its overexpression results in enhanced tolerance to multi-abiotic stress. Therefore, TaSnRK2.7 is a multifunctional regulatory factor in plants, and has the potential to be utilized in transgenic breeding to improve abiotic stress tolerance in crop plants.

Screening efficient siRNAs in vitro as the candidate genes for chicken anti-avian influenza virus H5N1 breeding.

The frequent disease outbreaks caused by avian influenza virus (AIV) not only affect the poultry industry but also pose a threat to human safety. To address the problem, RNA interference (RNAi) has recently been widely used as a potential antiviral approach. Transgenesis, in combination with RNAi to specifically inhibit AIV gene expression, has been proposed to make chickens resistant to avian influenza. For the transgenic breeding, screening the efficient siRNAs in vitro as the candidate genes is one of the most important tasks. Here, we combined an online search tool and a series of bioinformatics programs with a set of rules for designing the siRNAs targeting different mRNA regions of AIV H5N1 subtype. By this method we chose five rational siRNAs, constructed five U6 promoter-driven shRNA expression plasmids contained the siRNA genes, and used these to produce stably transfected Madin-Darby canine kidney (MDCK) cells. Data from virus titration, IFA, PUI-stained flow cytometry, real-time quantitative RT-PCR and DAS-ELISA analyses showed that all five stably transfected cell lines were effectively resistant to viral replication when exposed to 100 CCID50 of AIV, and we finally chose the most effective plasmids (pSi-604i and pSi-1597i) as the candidates for making the transgenic chickens. These findings provide baseline information for breeding transgenic chickens resistant to AIV in combination with RNAi.

Genetic transformation in Kappaphycus alvarezii using micro-particle bombardment: a potential strategy for germplasm improvement

This study investigated the delivery of a SV40 promoter driving lacZ gene into cells of Kappaphycus alvarezii using particle bombardment. Thallus pieces 0.5–0.8 mm in diameter and 1 cm in length were prepared as gene recipients. Bombardment parameters of 450 psi (rupture pressures) × 6 cm (particle travel distances), 650 psi × 6 cm, 1,100 psi × 6 cm and 1,100 psi × 9 cm were used. A significant increase in transformation efficiency from about 33% under the rupture pressure of 450 psi to 87% at 650 psi was observed in transformed thalli. Most of the positive cells appeared in epidermal cells bombarded at 450 psi, whereas positive signals were seen in both epidermal and medullary cells at 650 psi. No positive transient expression was detected at a bombardment of 1,100 psi, or in negative or blank controls. For the conditions tested, the best parameter was obtained at 650 psi at a distance of 6 cm. Thus, the strategy of taking vegetative thalli as recipients, using particle bombardment, and combining this with micro-propagation, together with developing an in vivo selectable marker, is a viable way to produce stable transformants, to eliminate chimeric expression, and to achieve transgenic breeding in K. alvarezii.

Development of Bt Rice by Molecular Marker-assisted Selection and Assays for Insect-Resistance

Rice germplasm 9311 and Fuhui 838 are both elite parent lines, particular for two and three-line indica hybrid rice in China, respectively. Two kinds of major lepidopteran pests in rice production, such as stemborers and leaffolders, cause severe yield loss in the most rice-producing countries. Bt toxic protein which are expressed by cry1Ac , cry1C* and cry2A* should be the most available methods to decrease the damage by these lepidopterans. To improve the rice resistance to insects, three Bt genes, i.e. cry1Ac , cry1C* and cry2A* , were introgressed to 9311 and Fuhui 838 from the donor parents, that are Minghui63 ( cry1C* ), Minghui63 ( cry2A* ), Minghui63 ( cry1Ac ) respectively by molecular maker-assisted selection. The results showed that the improved lines not only got greatly resistance to the pests, but also acquired good results of the agronomic traits. The improved lines would not only have potential application value but also can be used as bridge materials in rice transgenic breeding.

TaSnRK2.4, an SNF1-type serine/threonine protein kinase of wheat (Triticum aestivum L.), confers enhanced multistress tolerance in Arabidopsis

Osmotic stresses such as drought, salinity, and cold are major environmental factors that limit agricultural productivity worldwide. Protein phosphorylation/dephosphorylation are major signalling events induced by osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase2 family members play essential roles in response to hyperosmotic stresses in Arabidopsis, rice, and maize. In this study, the function of TaSnRK2.4 in drought, salt, and freezing stresses in Arabidopsis was characterized. A translational fusion protein of TaSnRK2.4 with green fluorescent protein showed subcellular localization in the cell membrane, cytoplasm, and nucleus. To examine the role of TaSnRK2.4 under various environmental stresses, transgenic Arabidopsis plants overexpressing wheat TaSnRK2.4 under control of the cauliflower mosaic virus 35S promoter were generated. Overexpression of TaSnRK2.4 resulted in delayed seedling establishment, longer primary roots, and higher yield under normal growing conditions. Transgenic Arabidopsis overexpressing TaSnRK2.4 had enhanced tolerance to drought, salt, and freezing stresses, which were simultaneously supported by physiological results, including decreased rate of water loss, enhanced higher relative water content, strengthened cell membrane stability, improved photosynthesis potential, and significantly increased osmotic potential. The results show that TaSnRK2.4 is involved in the regulation of enhanced osmotic potential, growth, and development under both normal and stress conditions, and imply that TaSnRK2.4 is a multifunctional regulatory factor in Arabidopsis. Since the overexpression of TaSnRK2.4 can significantly strengthen tolerance to drought, salt, and freezing stresses and does not retard the growth of transgenic Arabidopsis plants under well-watered conditions, TaSnRK2.4 could be utilized in transgenic breeding to improve abiotic stresses in crops.

Transgenic Breeding: Perspectives and Prospects

ABSTRACTTransgenic technology serves to introduce gene sequences for expression of a desired trait. Production of transgenic plants is reported in many crops, but commercialization is limited to a few selected crops, such as cotton (Gossypium hirsutum L.), corn (Zea mays L.), soybean [Glycine max (L.) Merr.], and canola (Brassica napus L. and B. rapa L.). This paper presents the sequential processes of transgenic event design, event selection, and “cleaning up” genetic background for forward breeding programs. Expression of the foreign gene cannot be viewed in isolation and is more complex than has been assumed because of the interaction of transgene with native genes. Variations among clones and within the progeny are observed, and hence all the clones are taken into account for evaluation. Plant breeding must be involved to move transgenes from transformable but agronomically unacceptable genotypes into elite breeding lines with two backcrosses. Production of transgenic plants in large numbers is difficult and laborious and requires large investments. It is worthwhile investing in parallel efforts to incorporate the transgene into improved plant material to satisfy commercial interests.

Abnormal maternal behavior, altered sociability, and impaired serotonin metabolism in Rai1-transgenic mice

Dup(17)(p11.2) syndrome, consisting of a spectrum of more than 20 clinical features, is associated with increased dosage of the retinoic acid induced 1 (RAI1) gene. We previously reported on the generation and evaluation of Rai1-overexpressing mice. Several phenotypes, including increased anxiety and hyperactivity, growth retardation, and altered motor and sensory coordination, were observed, recapitulating phenotypes observed in patients with 17p11.2 duplication. In addition, these mice have reduced reproductive fitness. In this study we expand investigations to identify possible neural correlates for increased Rai1 dosage. We analyzed Rai1-transgenic breeding data and evaluated maternal and social behaviors as potential causes for reduced litter size in Rai1 transgenics compared to wild-type controls. Abnormal maternal behavior, including delayed pup retrieval in the Rai1-transgenic dams compared to wild-type dams, was identified. Mendelian transmission of parental genotypes was also distorted in the pups from transgenic breeding. Furthermore, altered social behavior was observed in the male transgenic mice. Analysis of neurotransmitter levels from whole-brain lysates showed significantly impaired serotonin metabolism indicating a neuronal basis for behavioral modifications in these mice. Our study suggests an important role for Rai1 in the serotonin pathway in a dosage-dependent manner.