In order to optimize the overall efficiency of pronuclear microinjection, we designed experiments to: 1) test the best developmental stage for transferring injected embryos to obtain pregnancies and transgenic pups; 2) determine the optimum number of injected embryos transferred to obtain pregnancies and transgenic pups; 3) investigate whether addition of non-injected embryos with injected embryos increased pregnancy rate (PR) and transgenic pups; and 4) establish the time during pregnancy of highest embryonic or fetal loss. Mice (CD1; 3 to 4 wk old), were superovulated with 10 iu PMSG and 5 iu hCG 48 h apart. One-cell embryos were collected for microinjection 20 to 24 h after hCG. The gene used was the whey acidic protein promoter linked to a coding sequence of the human protein C gene (WAP-hPC). Embryos were cultured in CZB at 37°C in 5% CO₂ in air. All the live pups born and embryos and fetuses recovered were analyzed by the polymerase chain reaction to detect the presence of the transgene. Experiment one consisted of nine transfer treatments (TRT) which included all the combinations of three developmental stages (1-cell, 2-4 cell and morula/blastocyst) with three quantities of embryos per transfer (15-24, 25-34 and 35-44). Ten transfers were performed for each TRT. The highest PR and total pups born (TOTP) were obtained after transferring 25 to 34 2-4 cell embryos (PR=90% TOTP=3.5/ pregnancy). However, overall analysis indicated that the percentage of transgenic pups born (%TRS) was highest using 1-cell embryos [33.9%, 20.0% and 11.1% for 1-cell, 2-4 cell and morula/blastocyst (mor/bl), respectively]. The second experiment consisted of six transfer TRT: 20-0, 16-4, 12-8,30-0, 26-4, and 22-8 injected - non-injected embryos, respectively (10 transfers/TRT). Data showed that PR and TOTP can be improved by addition of non-injected embryos. However, the percentage of transgenic pups was significantly (p< .05) higher when all the embryos transferred were injected (53.6 % vs 46.4 % for transfers without and with non-manipulated embryos, respectively). Additionally, 30 embryos per transfer yielded a significantly higher percentage (p< .05) of transgenic pups than 20 embryos per transfer (67.9 % vs 32.1 % for 30 and 20 embryos per transfer, respectively). In experiment three 45 transfers of microinjected embryos were performed (30 embryos per transfer). Fifteen recipients were sacrificed on day 4, 12, and 18 of gestation. On each day all embryos and fetuses were counted and analyzed for the presence of the transgene. The percentage of transgenic embryos or fetuses was not statistically different at any recovery day (45.8%, 35.5%, and 34.6% for days 4, 12, and 18, respectively). However, the number of viable embryos at day 4 was significantly greater than the number of viable fetuses on days 12 or 18 (10 ± 1.1,,5.1 ± 1.6, and 2.4 ± 1.3 for days 4,,12 and 18, respectively). Collectively, the results indicate that: 1) transfer of 20 to 30 1-cell embryos was the best method to obtain transgenic mice, 2) addition of non-injected embryos decreased the number of transgenic pups obtained per pregnancy, and 3) although most of the embryonic losses after microinjection happen before day 4 of gestation, additional losses occurred between days 4 and 18 of pregnancy. / Ph. D.

Switchgrass (Panicum virgatum L.) is a candidate bioenergy crop. Somatic embryogenic (SE) calli are used for genetic transformation in switchgrass. A superior switchgrass line, HR8, was developed using recurrent tissue culture selection from cv. Alamo. HR8 SE calli were genetically transformable using Agrobacterium at an efficiency of ~12%. We used HR8 somatic embryogenic calli for genetic improvement of switchgrass. The lignin content of feedstock has been proposed as one key trait impacting biofuel production. 4-Coumarate: Coenzyme A ligase (4CL) is one of the key enzymes involved in the monolignol biosynthetic pathway. Two homologous 4CL genes, Pv4CL1 and Pv4CL2, were identified in switchgrass. Gene expression patterns and enzymatic activity assays suggested that Pv4CL1 is involved in monolignol biosynthesis. Stable transgenic plants were obtained with Pv4CL1 down-regulated. RNA interference of Pv4CL1 reduced extractable 4CL activity by 80%, leading to a reduction in lignin content with decreased guaiacyl unit composition. The transgenic plants had uncompromised biomass yield. After dilute acid pretreatment, the low lignin transgenic biomass had significantly increased cellulose hydrolysis (saccharification) efficiency for biofuel production. Erect leaf is a desirable trait to adjust the overall plant architecture to perceive more solar energy and thereby to increase the plant biomass production in a field population. We overexpressed an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (AtLOV1), in switchgrass. Surprisingly, AtLOV1 induced smaller leaf angle by changing morphologies of epidermal cells in the leaf collar region, affecting lignin content and monolignol composition, and also causing delayed flowering time in switchgrass. Global gene-expression analysis of AtLOV1 transgenic plants demonstrated an array of genes has altered expressions. Potential downstream genes involved in the pleiotropic phenotypic traits of the transgenic plants are discussed. / Ph. D.

Impatiens walleriana, commonly grown as a herbaceous annual, is susceptible to Impatiens Necrotic Spot Virus (INSV). A lack of resistant cultivars leaves growers with the sole option of destroying infected plants before INSV spreads throughout their entire crop. Therefore, the introduction of INSV resistant cultivars would have the potential to save Impatiens growers a substantial amount of money. Virus resistance has been successfully conveyed in several crops by insertion of pathogen DNA into the host plant. One method of generating transgenic plants involves the use of Agrobacterium-mediated gene transfer. A commonly used technique involves transformation of explant tissue and subsequent regeneration in vitro under aseptic conditions. However, prior to our research there was no regeneration protocol suitable for Agrobacterium-mediated transformation of Impatiens walleriana available. Herein we report the development of a new method for regeneration of Impatiens walleriana using cotyledonary node culture. Using this technique, four regeneration media amended with 1, 3, 5, or 7µM of thidiazuron were evaluated for their ability to induce de novo shoot production in cotyledonary node explants, and evaluated for number of shoots produced per explant. Results showed a significantly greater frequency of regeneration and number of shoots per explant using media amended with 1µM of thidiazuron. This technique has shown to be repeatable and is not susceptible to ploidy instability. Unfortunately, damage to the cotyledonary node explants during Agrobacterium inoculation and transfection prevented regeneration of transformed shoots in several attempts. However, transient GFP expression after transfection of shoot pads derived from cotyledonary nodes with Agrobacterium strain LBA 4404 containing plasmid pHB2829 with nptII and S-GFP was obtained, indicating the possibility for this regeneration protocol to derive stably transformed Impatiens with INSV resistance. / Master of Science

Tomato (Solanum lycopersicum) is a member of the Solanaceae family. In this research project tomato, more specifically the M82 cultivar was chosen as a model plant for Agrobacterium-mediated gene transfer by cotyledon inoculation. Our objective was to transform tomato with a T-DNA construct bearing a transposon from maize that can be used for mutagenesis when it transposes or moves around the genome of the tomato. The vector used is a two-component in-cis Ac-Ds system which needs a single transformation event. It was proved that it worked in Arabidopsis and rice according to Trijatmiko (2005). The construct consists of the BAR gene conferring resistance to herbicide Basta, hygromycin (HYG) gene conferring resistance to the antibiotic hygromycin and the green fluorescent protein (GFP) gene, which are driven by specific plant promoters. The selectable marker genes such as HYG and BAR were used to select the rare transformation events by making the transformed tomato tissue resistant to the toxic chemicals (antibiotic and herbicide) compared to the untransformed tissue in which growth was inhibited. The results described consist of developing a transformation protocol which enabled the production of transgenic tomato lines by the help of the antibiotic augmetin (amoxicillin/clavulanic acid). The transgenic lines were tested through polymerase chain reaction (PCR) and herbicide bioassays. / Master of Science

A simulation approach was used to determine the effects of multitrait selection on the correlations of sire direct and maternal predicted breeding values across environments. True and predicted direct and maternal breeding values (BV) of sires were simulated for sires evaluated independently in two different environments. Prediction error variances and covariances among direct and maternal BV within environments were required for the simulation. To obtain the necessary input parameters, a variety of MME coefficient matrices were created and inverted to inspect relationship among accuracies and correlations of prediction errors in sire evaluation models. An empirical prediction equation to predict the necessary prediction error covariances was obtained. Divergent, directional and random multitrait selection was then practiced using direct and maternal predicted BV as selection criteria. Samples of 40 sires were randomly obtained from each selected population. Observed correlations between direct and maternal predicted BV across environments were compared to expectations derived from univariate distribution theory. Selection definitely affected the expectations. However, the adjustment developed from univariate theory appeared to accommodate the effect of selection in these expectations. / Ph. D.

Thesis (PhD (Microbiology))--University of Stellenbosch, 2009. / ENGLISH ABSTRACT: Saccharomyces cerevisiae is the most commonly used organism in many fermentation-based industries including baking and the production of single cell proteins, biofuel and alcoholic beverages. In the wine industry, a consumer driven demand for new and improved products has focussed yeast research on developing strains with new qualities. Tremendous progress in the understanding of yeast genetics has promoted the development of yeast biotechnology and subsequently of genetically modified (GM) wine yeast strains. The potential benefits of such GM wine yeast are numerous, benefitting both wine makers and consumers. However, the safety considerations require intense evaluation before launching such strains into commercial production. Such assessments consider the possibility of the transfer of newly engineered DNA from the originally modified host to an unrelated organism. This process of horizontal gene transfer (HGT) creates a potential hazard in the use of such organisms. Although HGT has been extensively studied within the prokaryotic domain, there is an urgent need for similar studies on their eukaryotic counterparts. This study was therefore undertaken to help improve our understanding of this issue by investigating HGT in a model eukaryotic organism through a step-by-step approach. In a first step, this study attempted to determine whether large DNA fragments are released from fermenting wine yeast strains and, in a second step, to assess the stability of released DNA within such a fermenting background. The third step investigated in this study was to establish whether “free floating” DNA within this fermenting environment could be accepted and functionally expressed by the fermenting yeast cultures. Finally, whole plasmid transfer was also investigated as a unified event. Biofilms were also incorporated into this study as they constitute a possibly conducive environment for the observation of such HGT events. The results obtained during this study help to answer most of the above questions. Firstly, during an investigation into the possible release of large DNA fragments (>500 bp) from a GM commercial wine yeast strain (Parental strain: Vin13), no DNA could be detected within the fermenting background, suggesting that such DNA fragments were not released in large numbers. Secondly, the study revealed remarkable stability of free “floating DNA” under these fermentation conditions, identifying intact DNA of up to ~1kb in fermenting media for up to 62 days after it had been added. Thirdly, the data demonstrate the uptake and functional expression of spiked DNA by fermenting Vin13 cultures in grape must. Here, another interesting discovery was made, since it appears that the fermenting natural grape must favours DNA uptake when compared to synthetic must, suggesting the presence of carrier molecules. Additionally, we found that spiked plasmid DNA was not maintained as a circular unit, but that only the antibiotic resistance marker was maintained through genomic integration. Identification of the sites of integration showed the sites varied from one HGT event to the next, indicating that integration occurred through a process known as illegitimate recombination. Finally, we provide evidence for the direct transfer of whole plasmids between Vin13 strains. The overall outcome of this study is that HGT does indeed occur under the conditions investigated. To our knowledge, this is the first report of direct horizontal DNA transfer between organisms of the same species in eukaryotes. Furthermore, while the occurences of such events appears low in number, it cannot be assumed that HGT will not occur more frequently within an industrial scenario, making industrial scale studies similar to this one paramount before drawing further conclusions. / NO AFRIKAANS SUMMARY AVAILABLE

Thesis (MTech (Agriculture))--Cape Peninsula University Of Technology, 2016. / Bambara groundnut (Vigna subterranea), a dicotyledonous plant is a legume which has a potential to contribute to food security and nutrition. Protoplasts are naked plant cells lacking cell walls. Viable protoplasts are potentially totipotent. Therefore, when given the correct stimuli, each protoplast is capable, theoretically, of regenerating a new wall and undergoing repeated mitotic division to produce daughter cells from which fertile plants may be regenerated through the tissue culture process. Protoplast systems are valuable and versatile cell based systems that are useful in observing cellular processes and activities. In this study, the isolation of protoplast from the leaves of Bambara groundnut plant was extensively optimised. The factors affecting protoplast isolation considered in this study were ages of plant material, mannitol concentration, combinations and concentrations of enzymes and duration of incubation. Effects of ages of Bambara groundnut plant (4, 6, 8, 10 weeks), molarities of mannitol (0.4 M, 0.5 M. 0.6 M and 0.7 M), concentration and combination of enzymes (1%, 2% and 4% cellulase, 0.5% and 1% macerozyme and, 0.5% and 1% pectinase) at different incubation duration (4, 18, 24, 42 hours) were investigated. Overall, it can be deduced from this study that the optimal protoplast yield (4.6 ± 0.14×105ml-1/gFW) and viability (86.5 ± 2.12%) were achieved by digesting the leaves of four week old Bambara groundnut plant with 2% cellulase and 0.5 % macerozyme with 0.5M mannitol for 18 hours. Freshly isolated protoplasts were then cultured at different densities of 1 × 104 - 2 ×106 protoplasts/ml using MS in three different culture (Liquid, agar and agarose bead) methods. First cell division was observed only in liquid medium. With several attempts, no division was achieved in the agar and agarose bead methods, division also did not progress in the liquid medium and hence, plant regeneration from Bambara groundnut protoplasts could not be achieved in this study. Consequently, a further study is underway to compare the proteomic profiles of freshly isolated protoplasts and cultured protoplasts in order to gain insights into the expression of proteins that could perhaps be contributing to the difficulty in regenerating Bambara groundnut plant through protoplast technology. The present study is novel because it is the first study to optimise the various factors that could affect protoplast isolation from the leaves of Bambara groundnut and thus developed an efficient protocol for protoplasts isolation from leaves of Bambara groundnut for cell manipulation studies.

A transformação genética é uma alternativa ao melhoramento convencional de citros que permite a modificação de genótipos pela introdução de um ou mais genes oriundos de organismos semelhantes ou filogeneticamente distantes do hospedeiro. Os genes transferidos para espécies de interesse devem ser controlados por promotores, os quais regulam a expressão gênica de forma temporal, espacial e na magnitude desejada. Na maioria dos casos, os genes são expressos de forma constitutiva utilizando o promotor CaMV35S isolado do Vírus do Mosaico da Couve Flor. No entanto, o desenvolvimento de novas abordagens de transformação de plantas (cisgenia e intragenia), que fazem o uso de genes e sequências regulatórias derivadas da mesma espécie ou espécies relacionadas, requer a disponibilidade de elementos genéticos, incluindo promotores constitutivos, isolados de citros. Assim, o objetivo do trabalho foi clonar e caracterizar promotores constitutivos de Citrus sinensis. Para isso, a região promotora dos genes Fator de elongação 1-&alpha; (CsEF1), Gliceraldeido-3-fosfato-desidrogenase C2 (CsGAPC2) e Cyclofilina (CsCYC) foi isolada e avaliados pela fusão ao gene repórter uidA. A funcionalidade dos três promotores foi confirmada por ensaio histoquímico da atividade GUS em folhas, caules e raízes de plantas transgênicas de citros cv. \'Hamlin\'. A análise de RT-qPCR mostra que a expressão do gene uidA sob controle dos promotores CsCYC, CsGAPC2 e CsEF1 correspondeu a uma atividade aproximada de 64%, 58% e 47%, respectivamente em comparação com o promotor CaMV35S. A análise in silico dos promotores CsGAPC2, CsCYC e CsEF1 mostra que a atividade de cada um é controlada por uma série de putativos elementos cis-regulatórios. A sequência completa e versões truncadas originadas a partir de deleções em cada promotor foram fundidas ao gene uidA e analisadas em plantas transgênicas de Nicotiana benthamiana pelo ensaio histoquímico e fluorimétrico da atividade GUS. As análises de deleções não causaram perda de função dos promotores em estudo, mas afetaram a expressão gênica nos promotores CsGAPC2 e CsEF1. Os promotores isolados representam bons candidatos a serem utilizados em trabalhos de transformação genética de citros. / Genetic transformation is an alternative to citros conventional breeding that allows the modification of genotypes by the introduction of one or more genes derived from different organisms that can not be crossed by natural means. The transferred genes to the species of interest are controlled by promoters, which regulate a gene expression temporally, spatially and in the desired magnitude. In most cases, the introduced genes have been constitutively expressed using the CaMV35S promoter obtained from the cauliflower mosaic virus. However, the development of novel plant transformation approaches (cisgenesis and intragenesis) imply the use of genetic material from the same species or from closely related species capable of sexual hybridization, which requires the isolation of genetic elements, including citros constitutive promoters. The objective of this study was clone and characterize Citrus sinensis constitutive promoters. For this, the promoter region of the genes Elongation Factor 1-&alpha; (CsEF1), Glyceraldehyde-3-phosphate dehydrogenase C2 (CsGAPC2) and Cyclofiline (CsCYC) was isolated and evaluated by fusion to the uidA reporter gene. The functionality of three promoter was confirmed by histochemical GUS assay in leaves, stems and roots of transgenic citrus plants cv. \'Hamlin\'. RT-qPCR analysis revealed that uidA gene expression under control of the CsCYC, CsGAPC2 and CsEF1 promoters was approximately 64%, 58% and 47% expression compared with the CaMV35S promoter. In silico analysis of the CsGAPC2, CsCYC and CsEF1 promoters displays their activity is controlled by a series of putative cis-regulatory elements. The full length promoter and truncated versions originated from deletions in promoters sequences were fused to the uidA gene and analyzed in Nicotiana benthamiana transgenic plants by histochemical and fluorimetric GUS assay. Deletion analysis did not cause loss of function on any of the promoters, but affected the gene expression on CsGAPC2 and CsEF1 truncated versions. The isolated promoters represent good candidates to be used in citros genetic transformation.

O vírus da tristeza dos citros (CTV) é uma das maiores ameaças à citricultura mundial. No Brasil, mesmo com a pré-imunização e com a substituição de porta-enxertos, estirpes fortes de CTV ainda causam prejuízos consideráveis. Com o aparecimento da Morte Súbita dos Citros em 1999 e a possível relação desta doença com o CTV, este vírus voltou a figurar como patógeno de importância no cenário da citricultura brasileira. Uma das possíveis soluções para o controle de viroses em fruteiras é a obtenção de plantas transgênicas resistentes ou imunes. O objetivo deste trabalho foi avaliar a resistência ao CTV de plantas transgênicas de laranja \'Hamlin\' contendo três construções gênicas oriundas de seqüências do genoma do CTV. Estas construções gênicas visaram ativar rotas de RNAi (hairpin da capa protéica e seqüência conservada antisenso do CTV) e mecanismos de defesa relacionados à expressão da capa protéica do CTV. As plantas transgênicas foram desafiadas com uma estirpe fraca de CTV (CTV-IAC) por meio de borbulhas e pulgões pretos (Toxoptera citricida Kirkaldy) contendo o vírus. A avaliação da resistênica à replicação viral foi feita por análises de ELISA. As plantas transgênicas foram consideradas não resistentes à infecção e translocação viral quando inoculadas com borbulhas. Entretanto algumas plantas mostraram retardamento da infecção. Não foi possível determinar se houve resistência à transmissão de CTV por pulgões já que a técnica utilizada não foi capaz de infectar os controles de maneira uniforme. / The Citrus tristeza virus (CTV) is one of the greatest threats to the citrus industry worldwide. In Brazil, CTV continues to cause damage through strong strains despite the use of techniques like cross-protection and substitution of intolerant rootstocks. With the appearance and spread of the Citrus Sudden Death disease in 1999 and its possible relation to CTV, this virus was again among important pathogens within the Brazilian citrus industry. One of the possible solutions for controlling virus diseases in fruit crops is the development of immune or resistant transgenic plants. The objective of this work was to evaluate the resistance to CTV of transgenic \'Hamlin\' sweet orange plants containing three transgenic constructs obtained from CTV genomic sequences. The genetic constructs used aimed to activate RNAi defense routes (coat protein hairpin and a conserved sequence from CTV) and resistance mechanisms related to the coat protein expression. The transgenic plants were challenged with a weak strain of CTV, CTV-IAC, by bud and aphid (Toxoptera citricida Kirkaldy) inoculation. The evaluation of viral replication was done by ELISA analysis. The transgenic plants were considered susceptible to viral replication and translocation when bud inoculated. However, a few plants showed retardation of infection. It was not possible to determine resistance in the aphid transmission assay since the controls were not uniformly inoculated.

A contaminação de dornas por leveduras selvagens pode prejudicar o rendimento e a produtividade da produção industrial de etanol, inexistindo métodos eficientes para o controle deste tipo de contaminação. Facilidade, rapidez e o baixo custo seriam características desejáveis para o controle de leveduras que eventualmente contaminam o processo de fermentação. Com este objetivo, TAVARES (1989) desenvolveu um processo de produção de etanol com o controle de leveduras contaminantes, que utiliza um híbrido M606 de Saccharomyces cerevisiae de alta produtividade e resistente a nistatina, um antifúngico de amplo espectro que pode ser usado para garantir a pureza genética do inóculo industrial. Para facilitar a identificação de leveduras GOMES et al. (2000), utilizaram a propriedade de fluorescência expressada pelo gene GFP \"Green Fluorescent Protein\" sob o controle do promotor da da ADH2 em baixas concentrações de glicose. Associar estas duas propriedades em uma levedura industrial permitiria manter a pureza do inóculo e facilitaria a sua identificação. Com este objetivo, foram efetuados experimentos de transformação genética do híbrido M606 com o plasmídio pYGFP3 construído por Gomes et al (2000), sem obter o sucesso desejado. Por isto, como passo inicial para obtenção de ambas propriedades em híbrido altamente produtivo, optou-se pela metodologia de cruzamentos entre uma linhagem segregante haplóide resistente a nistatina M606-2c(n) e a linhagem X2904- GFP3 portadora do plasmídio pYGFP3. Alguns híbridos deste cruzamento natural foram selecionados, para posterior avaliação do nível de resistência à nistatina, da estabilidade do plasmídio pYGFP3 e da capacidade fermentativa. Verificou-se que os híbridos selecionados (P16, P34 e P42) foram capazes de crescer numa concentração de 10mg.L-1 de nistatina. Contudo, detectou-se instabilidade do plasmídio pYGFP3 em todos os híbridos selecionados. Os híbridos P34 e P42 demonstraram uma capacidade fermentativa inferior às linhagens controle, o que pode ser explicado pela fragilidade do sistema de membranas decorrente da natureza da resistência à nistatina. O híbrido P16 não apresentou diferenças na capacidade de fermentação em relação as linhagens controle. Apesar de obter híbridos resistentes a nistatina expressando o gene GFP3, verifica-se que há necessidade de modificar o plasmídio pYGFP3 tornando-o integrativo no genoma da levedura, para permitir a estabilidade do gene GFP3. / Vats contamination by wild yeasts can harm the efficiency and the productivity of ethanol industrial production and there are not efficient methods to control this type of contamination. Easiness, speed and the low cost would be desirable characteristics for the control of yeasts that eventually contaminate the fermentation process. With this aim, TAVARES (1989) developed an ethanol production process with the control of spoilage yeasts contaminants, that uses a Saccharomyces cerevisiae hybrid M606, of high productivity and resistant to the nystatin. It´s a wide spectrum antifungal that can be used to guarantee the genetic purity of the industrial inoculum. To facilitate the yeasts identification, GOMES et al. (2000) used the fluorescence property expressed by the GFP gene \"Green Fluorescent Protein\", that is controlled by the promoter of ADH2 in a low glucose concentrations. The association of these two properties in an industrial yeast strain would allow to maintain the purity of the inoculum and it would facilitate its identification. With this aim, an assay of genetic transformation of hybrid M606 with pYGFP3 plasmid built by Gomes et al (2000) was made, but it had not success. Therefore, as initial step for obtaining of both properties in hybrid highly productive, It was opted for the methodology of crossings between a nystatin resistant haploid segregant strain M606-2c(n) and the strain X2904-GFP3(n) harboring of the plasmid pYGFP3. Some hybrids were selected of this natural crossing, with subsequent evaluation of nystatin resistance level, stability of the pYGFP3 plasmid and fermentative capacity. The selected hybrids (P16, P34 and P42) were capable to grow in a concentration of 10mg.nystatin L-1. However, plasmidial instability of the pYGFP3 was detected in all the selected hybrids. The hybrids P34 and P42 showed a smaller fermentative capacity than control strain, which can be explained by the fragility of the membranes system due to the nature of the resistance to the nystatin. The hybrid P16 did not showed difference in the fermentative capacity to the strain control. In spite of obtaining resistant hybrid to the nystatin expressing the GFP3 gene, there is a need to modify the pYGFP3 plasmid, turning it integrative in the yeast genome to allow the gene stability GFP3.