Stage-specific Promoters Research Articles

De novo transcriptome assembly of mouse male germ cells reveals novel genes, stage-specific bidirectional promoter activity, and noncoding RNA expression.

In mammals, the adult testis is the tissue with the highest diversity in gene expression. Much of that diversity is attributed to germ cells, primarily meiotic spermatocytes and postmeiotic haploid spermatids. Exploiting a newly developed cell purification method, we profiled the transcriptomes of such postmitotic germ cells of mice. We used a de novo transcriptome assembly approach and identified thousands of novel expressed transcripts characterized by features distinct from those of known genes. Novel loci tend to be short in length, monoexonic, and lowly expressed. Most novel genes have arisen recently in evolutionary time and possess low coding potential. Nonetheless, we identify several novel protein-coding genes harboring open reading frames that encode proteins containing matches to conserved protein domains. Analysis of mass-spectrometry data from adult mouse testes confirms protein production from several of these novel genes. We also examine overlap between transcripts and repetitive elements. We find that although distinct families of repeats are expressed with differing temporal dynamics during spermatogenesis, we do not observe a general mode of regulation wherein repeats drive expression of nonrepetitive sequences in a cell type-specific manner. Finally, we observe many fairly long antisense transcripts originating from canonical gene promoters, pointing to pervasive bidirectional promoter activity during spermatogenesis that is distinct and more frequent compared with somatic cells.

The apicoplast is important for the viability and persistence of Toxoplasma gondii bradyzoites

Toxoplasma gondii is responsible for toxoplasmosis, a disease that can be serious when contracted during pregnancy, but can also be a threat for immunocompromised individuals. Acute infection is associated with the tachyzoite form that spreads rapidly within the host. However, under stress conditions, some parasites can differentiate into cyst-forming bradyzoites, residing mainly in the central nervous system, retina and muscle. Because this latent form of the parasite is resistant to all currently available treatments, and is central to persistence and transmission of the parasite, specific therapeutic strategies targeting this developmental stage need to be found. T. gondii contains a plastid of endosymbiotic origin called the apicoplast, which is an appealing drug target because it is essential for tachyzoite viability and contains several key metabolic pathways that are largely absent from the mammalian host. Its function in bradyzoites, however, is unknown. Our objective was thus to study the contribution of the apicoplast to the viability and persistence of bradyzoites during chronic toxoplasmosis. We have used complementary strategies based on stage-specific promoters to generate conditional bradyzoite mutants of essential apicoplast genes. Our results show that specifically targeting the apicoplast in both in vitro or in vivo-differentiated bradyzoites leads to a loss of long-term bradyzoite viability, highlighting the importance of this organelle for this developmental stage. This validates the apicoplast as a potential area to look for therapeutic targets in bradyzoites, with the aim to interfere with this currently incurable parasite stage.

Evidence of high-efficiency cross fertilization in Eimeria acervulina revealed using two lines of transgenic parasites

Eimeria species are apicomplexan parasites with a direct life cycle consisting of a replicative phase involving multiple rounds of asexual replication in the intestine or other organs including kidneys, liver, and gallbladder, depending on the species, followed by a sexual phase or gamogony involving the development and fertilization of gametes, an essential process for Eimeria transmission. Recent advances in the genetic manipulation of these parasites made it possible to conduct genetic crosses combined with genomic approaches to elucidate the genetic determinants of Eimeria development, virulence, drug resistance, and immune evasion. Here, we employed genetic techniques to generate two transgenic Eimeria acervulina lines, EaGAM56 and EaHAP2, each expressing two unique fluorescent proteins, with one controlled by a constitutive promotor for cross-efficiency analysis and the other by a male or female gametocyte stage-specific promoter to observe sexual development. The expression of fluorescent proteins in the transgenic lines was analyzed in different developmental stages of the E. acervulina life cycle by immunoblotting and by examination of frozen sections using fluorescence microscopy. The effect of infective doses on cross-fertilization was further investigated by conducting several genetic crosses between the two transgenic lines at different doses and ratios. Two transgenic lines expressing constitutive and gametocyte-specific fluorescence proteins were generated and characterized. These transgenic parasites display synchronous development in chickens, comparable with that of the wild type. Genetic crosses between the two transgenic parasites showed that a high rate of oocysts co-expressing the two reporters could be achieved following inoculation with high doses of infective oocysts. We further showed that the proportion of co-transfected oocysts can be modulated by altering the ratio of the transgenic parental lines. Higher infective doses and similar numbers of functional gametocytes from the parents increase the rate of cross-fertilization. Our data highlight the usefulness of genetic manipulation and fluorescently-labeled transgenic gametocytes as tools to study Eimeria development and to elucidate the factors that modulate sexual development. This work sets the stage for the implementation of novel approaches to investigate other aspects of Eimeria pathogenesis, virulence, and drug susceptibility and resistance.

Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

TSS-seq of Toxoplasma gondii sporozoites revealed a novel motif in stage-specific promoters.

Toxoplasma gondii is one of the most common zoonotic protozoan parasites. It has three major infectious stages: rapidly multiplying tachyzoites (Tz), slowly replicating bradyzoites (Bz) and a resting/free-living stage, sporozoites (Sz). The regulatory mechanisms governing stage-specific gene expression are not fully understood. Few transcriptional start sites (TSS) are known for Sz. In this study, we obtained TSS of Sz using an oligo-capping method and RNA-seq analysis. We identified 1,043,503 TSS in the Sz transcriptome. These defined 38,973 TSS clusters, of which, 11,925 were expressed in Sz and 1535 TSS differentially expressed in Sz. Based on these data, we defined promoter regions and novel sporozoite stage-specific motifs using MEME. TGTANNTACA was distributed around -55 to -75 regions from each TSS. Interestingly, the same motif was reported in another apicomplexan, Plasmodium berghei, as a cis-element of female-specific gametocyte genes, implying the presence of common regulatory machinery. Further comparative analysis should better define the distribution and function of these elements in other members of this important parasitic phylum.

A toolbox for conditional control of gene expression in apicomplexan parasites.

Apicomplexan parasites encompass diverse pathogens for humans and animals, including the causative agents of malaria and toxoplasmosis, Plasmodium spp. and Toxoplasma gondii. Genetic manipulation of these parasites has become central to explore parasite biology, unravel gene function and identify new targets for therapeutic strategies. Tremendous progress has been achieved over the past years with the advent of next generation sequencing and powerful genome editing methods. In particular, various methods for conditional gene expression have been developed in both Plasmodium and Toxoplasma to knockout or knockdown essential genes, or for inducible expression of master developmental regulators or mutant versions of proteins. Conditional gene expression can be achieved at three distinct levels. At the DNA level, inducible site‐specific recombinases allow conditional genome editing. At the RNA level, regulation can be achieved during transcription, using stage‐specific or regulatable promoters, or post‐transcriptionally through alteration of mRNA stability or translation. At the protein level, several systems have been developed for inducible degradation or displacement of a protein of interest. In this review, we provide an overview of current systems for conditional control of gene expression in Plasmodium and Toxoplasma parasites, highlighting the advantages and limitations of each approach.

The transcriptome landscapes of citrus leaf in different developmental stages.

The temporal expression profiles of citrus leaves explain the sink-source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses of mature and immature leaves to biotic stress such as citrus canker and Asian citrus psyllid (Diaphorina citri). Citrus is an important fruit crop worldwide. Different developmental stages of citrus leaves are associated with distinct features, such as differences in susceptibilities to pathogens and insects, as well as photosynthetic capacity. Here, we investigated the mechanisms underlying these distinctions by comparing the gene expression profiles of mature and immature citrus leaves. Immature (stages V3 and V4), transition (stage V5), and mature (stage V6) Citrus sinensis leaves were chosen for RNA-seq analyses. Carbohydrate biosynthesis, photosynthesis, starch biosynthesis, and disaccharide metabolic processes were enriched among the upregulated differentially expressed genes (DEGs) in the V5 and V6 stages compared with that in the V3 and V4 stages. Glucose level was found to be higher in V5 and V6 than in V3 and V4. Among the four stages, the largest number of DEGs between contiguous stages were identified between V5 and V4, consistent with a change from sink to source, as well as with the sucrose and starch quantification data. The differential expression profiles related to cell wall synthesis, secondary metabolites such as flavonoids and terpenoids, amino acid biosynthesis, and immunity between immature and mature leaves may contribute to their different responses to Asian citrus psyllid infestation. The expression data suggested that both the constitutive and induced gene expression of immunity-related genes plays important roles in the greater resistance of mature leaves against Xanthomonas citri compared with immature leaves. The gene expression profiles in the different stages can help identify stage-specific promoters for the manipulation of the expression of citrus traits according to the stage. The temporal expression profiles explain the sink-source transition of immature leaves to mature leaves and provide knowledge regarding the differential responses to biotic stress.

Review A Short Review of Promoters of Cotton Fibre Genes: Strength and Tissue Specificity


 Promoters are of great importance for controlling gene expression. There are various regulatory regions within promoters which are crucial for expression specificity. Depending upon mode of expression, promoters can be divided into constitutive, tissue/developmental stage specific or inducible promoters. For expression of certain gene, there is interplay of cis acting element located on promoter and transcription factors. Cotton fibre promoters are of great importance in biotechnology as these may be used to express fibre genes in cotton fibres specifically. Although most of cotton fibre gene promoters show expression in fibres but some of them also exhibit expression in various other organs like trichomes, pedicles and vascular tissues. Due to difficulties in somatic embryogenesis in cotton, these promoters are mostly studied in heterologous systems like Arabidopsis and tobacco. In this article, expression pattern of some cotton fibre genes promoters have been reviewed.
 

In-Depth Two-Stage Transcriptional Reprogramming and Evolutionary Engineering of Saccharomyces cerevisiae for Efficient Bioethanol Production from Xylose with Acetate.

In order to achieve rapid xylose utilization in the presence of acetate, improved yeast strains were engineered for higher bioethanol production. First, a six-gene cluster, including XYL1/XYL2/XKS1/TAL1/PYK1/MGT05196, was generated by using an in-depth two-stage (glucose and xylose) transcription reprogramming strategy in an evolutionary adapted strain of CE7, resulting in two improved engineered strains WXY46 and WXY53. Through a combined screening of xylose and glucose stage-specific promoters between tricarboxylic acid (TCA)/HSP and constitutive types, respectively, WXY46 with the constitutive promoters showed a much higher ethanol yield than that of WXY53 with the TCA/HSP promoters. Second, an optimized strain WXY74 was obtained by using more copies of a six-gene cluster, which resulted in a higher ethanol yield of 0.500 g/g total sugars with acetate conditions. At last, simultaneous saccharification and co-fermentation were performed by using the evolved WXY74 strain, which produced 58.4 g/L of ethanol from wheat straw waste and outperformed previous haploid XR-XDH strains.

Novel promoters for constitutive and inducible expression of transgenes in the diatom Phaeodactylum tricornutum under varied nitrate availability

Recent developments in the genetic engineering of biotechnologically important microalgae—among them the diatom Phaeodactylum tricornutum—promise rapid progress in strain improvements for producing various valuable commodities. Nitrogen (N) depletion and starvation are commonly employed strategies to induce or enhance the formation of secondary carotenoids and storage lipids in microalgal biotechnology. The manipulation of gene expression for improving or rerouting the metabolic pathways requires the development of suitable stage-specific promoters to drive gene expression under desired conditions. Here, we describe the isolation, cloning, and characterization of novel P. tricornutum promoters that are either constitutively active in the presence or absence of nitrate as the sole N source, or upregulated under N starvation. An enhanced green fluorescent protein (eGFP), fused to a plasma membrane-anchoring sequence at the C terminus, was expressed under the control of several endogenous promoters, which were chosen based on previously reported gene expression levels under nitrate-limited or deprived conditions. Among the four promoters tested in this work, the ammonium transporter gene (AMT, PHATRDRAFT_27877) promoter strongly induced eGFP accumulation under N starvation, and the purine permease gene (PUP, PHATRDRAFT_52619) promoter strongly and constitutively drove eGFP accumulation, regardless of nitrate availability. The Act2 (actin-like 2, PHATRDRAFT_29136) and previously characterized DGAT1 gene promoters had relatively weak constitutive and late-inducible expression, respectively, under N starvation. The fucoxanthin-chlorophyll a/c-binding protein gene (fcpA) promoter used as a control was downregulated under N starvation. A comparison of quantitative real-time PCR gene expression data with western blot results suggested that controlled transcriptional regulation is important for efficient transgenic protein accumulation under N starvation. The results are of interest for the expression of transgenes in P. tricornutum for engineering oil production capacity and accumulating recombinant proteins.

Regulatory elements driving the expression of skeletal lineage reporters differ during bone development and adulthood

To improve bone healing or regeneration more insight in the fate and role of the different skeletal cell types is required. Mouse models for fate mapping and lineage tracing of skeletal cells, using stage-specific promoters, have advanced our understanding of bone development, a process that is largely recapitulated during bone repair. However, validation of these models is often only performed during development, whereas proof of the activity and specificity of the used promoters during the bone regenerative process is limited. Here, we show that the regulatory elements of the 6kb collagen type II promoter are not adequate to drive gene expression during bone repair. Similarly, the 2.3kb promoter of collagen type I lacks activity in adult mice, but the 3.2kb promoter is suitable. Furthermore, Cre-mediated fate mapping allows the visualization of progeny, but this label retention may hinder to distinguish these cells from ones with active expression of the marker at later time points. Together, our results show that the lineage-specific regulatory elements driving gene expression during bone development differ from those required later in life and during bone repair, and justify validation of lineage-specific cell tracing and gene silencing strategies during fracture healing and bone regenerative applications.

Capturing in vivo RNA transcriptional dynamics from the malaria parasite Plasmodium falciparum.

To capture the transcriptional dynamics within proliferating cells, methods to differentiate nascent transcription from preexisting mRNAs are desired. One approach is to label newly synthesized mRNA transcripts in vivo through the incorporation of modified pyrimidines. However, the human malaria parasite, Plasmodium falciparum, is incapable of pyrimidine salvage for mRNA biogenesis. To capture cellular mRNA dynamics during Plasmodium development, we engineered parasites that can salvage pyrimidines through the expression of a single bifunctional yeast fusion gene, cytosine deaminase/uracil phosphoribosyltransferase (FCU). We show that expression of FCU allows for the direct incorporation of thiol-modified pyrimidines into nascent mRNAs. Using developmental stage-specific promoters to express FCU-GFP enables the biosynthetic capture and in-depth analysis of mRNA dynamics from subpopulations of cells undergoing differentiation. We demonstrate the utility of this method by examining the transcriptional dynamics of the sexual gametocyte stage transition, a process that is essential to malaria transmission between hosts. Using the pfs16 gametocyte-specific promoter to express FCU-GFP in 3D7 parasites, we found that sexual stage commitment is governed by transcriptional reprogramming and stabilization of a subset of essential gametocyte transcripts. We also measured mRNA dynamics in F12 gametocyte-deficient parasites and demonstrate that the transcriptional program required for sexual commitment and maturation is initiated but likely aborted due to the absence of the PfAP2-G transcriptional regulator and a lack of gametocyte-specific mRNA stabilization. Biosynthetic labeling of Plasmodium mRNAs is incredibly versatile, can be used to measure transcriptional dynamics at any stage of parasite development, and will allow for future applications to comprehensively measure RNA-protein interactions in the malaria parasite.

Identification of the MUC2 Promoter as a Strong Promoter for Intestinal Gene Expression through Generation of Transgenic Quail Expressing GFP in Gut Epithelial Cells.

Identification of tissue- and stage-specific gene promoters is valuable for delineating the functional roles of specific genes in genetically engineered animals. Here, through the comparison of gene expression in different tissues by analysis of a microarray database, the intestinal specificity of mucin 2 (MUC2) expression was identified in mice and humans, and further confirmed in chickens by RT-PCR (reverse transcription-PCR) analysis. An analysis of cis-acting elements in avian MUC2 gene promoters revealed conservation of binding sites, within a 2.9 kb proximal promoter region, for transcription factors such as caudal type homeobox 2 (CDX2), GATA binding protein 4 (GATA4), hepatocyte nuclear factor 4 α (HNF4A), and transcription factor 4 (TCF4) that are important for maintaining intestinal homeostasis and functional integrity. By generating transgenic quail, we demonstrated that the 2.9 kb chicken MUC2 promoter could drive green fluorescent protein (GFP) reporter expression exclusively in the small intestine, large intestine, and ceca. Fluorescence image analysis further revealed GFP expression in intestine epithelial cells. The GFP expression was barely detectable in the embryonic intestine, but increased during post-hatch development. The spatiotemporal expression pattern of the reporter gene confirmed that the 2.9 kb MUC2 promoter could retain the regulatory element to drive expression of target genes in intestinal tissues after hatching. This new transgene expression system, using the MUC2 promoter, will provide a new method of overexpressing target genes to study gene function in the avian intestine.

Rapid Generation of Marker-Free P. falciparum Fluorescent Reporter Lines Using Modified CRISPR/Cas9 Constructs and Selection Protocol.

The CRISPR/Cas9 system is a powerful genome editing technique employed in a wide variety of organisms including recently the human malaria parasite, P. falciparum. Here we report on further improvements to the CRISPR/Cas9 transfection constructs and selection protocol to more rapidly modify the P. falciparum genome and to introduce transgenes into the parasite genome without the inclusion of drug-selectable marker genes. This method was used to stably integrate the gene encoding GFP into the P. falciparum genome under the control of promoters of three different Plasmodium genes (calmodulin, gapdh and hsp70). These genes were selected as they are highly transcribed in blood stages. We show that the three reporter parasite lines generated in this study (GFP@cam, GFP@gapdh and GFP@hsp70) have in vitro blood stage growth kinetics and drug-sensitivity profiles comparable to the parental P. falciparum (NF54) wild-type line. Both asexual and sexual blood stages of the three reporter lines expressed GFP-fluorescence with GFP@hsp70 having the highest fluorescent intensity in schizont stages as shown by flow cytometry analysis of GFP-fluorescence intensity. The improved CRISPR/Cas9 constructs/protocol will aid in the rapid generation of transgenic and modified P. falciparum parasites, including those expressing different reporters proteins under different (stage specific) promoters.

Fruit preferential activity of the tomato RIP1 gene promoter in transgenic tomato and Arabidopsis

Isolation and functional characterization of tissue- and stage-specific gene promoters is beneficial for genetic improvement of economically important crops. Here, we have characterized a putative promoter of a ripening-induced gene RIP1 (Ripening induced protein 1) in tomato. Quantification of the transcript level of RIP1 showed that its expression is fruit preferential, with maximum accumulation in red ripe fruits. To test the promoter activity, we made a reporter construct by cloning 1450bp putative RIP1 promoter driving the GUS (ß-glucuronidase) gene expression and generated stable transgenic lines in tomato and Arabidopsis. Histochemical and fluorometric assays validated the fruit-specific expression of RIP1 as the highest GUS activity was found in red ripe tomatoes. Similarly, we detected high levels of GUS activity in the siliques of Arabidopsis. On the contrary, weak GUS activity was found in the flower buds in both tomato and Arabidopsis. To characterize the specific regions of the RIP1 promoter that might be essential for its maximum activity and specificity in fruits, we made stable transgenic lines of tomato and Arabidopsis with 5'-deletion constructs. Characterization of these transgenic plants showed that the full length promoter is essential for its function. Overall, we report the identification and characterization of a ripening-induced promoter of tomato, which would be useful for the controlled manipulation of the ripening-related agronomic traits in genetic manipulation studies in future.

Comparative Plasmodium gene overexpression reveals distinct perturbation of sporozoite transmission by profilin.

Plasmodium relies on actin-based motility to migrate from the site of infection and invade target cells. Using a substrate-dependent gliding locomotion, sporozoites are able to move at fast speed (1-3 μm/s). This motility relies on a minimal set of actin regulatory proteins and occurs in the absence of detectable filamentous actin (F-actin). Here we report an overexpression strategy to investigate whether perturbations of F-actin steady-state levels affect gliding locomotion and host invasion. We selected two vital Plasmodium berghei G-actin-binding proteins, C-CAP and profilin, in combination with three stage-specific promoters and mapped the phenotypes afforded by overexpression in all three extracellular motile stages. We show that in merozoites and ookinetes, additional expression does not impair life cycle progression. In marked contrast, overexpression of C-CAP and profilin in sporozoites impairs circular gliding motility and salivary gland invasion. The propensity for productive motility correlates with actin accumulation at the parasite tip, as revealed by combinations of an actin-stabilizing drug and transgenic parasites. Strong expression of profilin, but not C-CAP, resulted in complete life cycle arrest. Comparative overexpression is an alternative experimental genetic strategy to study essential genes and reveals effects of regulatory imbalances that are not uncovered from deletion-mutant phenotyping.

Development of dual fluorescent stage specific reporter strain of Toxoplasma gondii to follow tachyzoite and bradyzoite development in vitro and in vivo

Toxoplasma gondii is a protozoan that infects 30% of humans as intermediate hosts. T Sexual reproduction can occur only within the intestinal tract of felines, however, infection in other mammals and birds is associated with asexual replication and interconversion between the tachyzoite and bradyzoite stages. Bradyzoites are slow growing forms found in tissue cysts in latent infection. Recently, our group described the biological behavior of the EGS strain that forms thick walled cysts spontaneously in tissue culture, constituting a useful tool for examining the developmental biology of T. gondii. To further improve the usefulness of this model, we constructed genetically modified EGS parasites that express fluorescent tags under the control of stage specific promoters. The promoter regions for SAG-1 (tachyzoite specific), BAG-1 and LDH-2 (bradyzoite specific) were amplified by PCR and plasmids were constructed with mCherry (redT) and sfGFP (greenB) sequences, respectively. Strains of parasites were selected using FACS to arrive at single fluorescent and dual fluorescent strains of EGS expressing tags in a stage specific manner. In cell cultures, vacuoles labeled by immunofluorescence assay using anti-CST-1 a marker for T. gondii cyst wall contained parasites that were positive for BAG1-GFP and negative for SAG1-mCherry. Tachyzoites and bradyzoites harvested from the mice expressed stage specific mCherry and GFP proteins, respectively. These new dual fluorescent transgenic EGS strains are a promising tool to elucidate the mechanisms of T. gondii differentiation both in vitro and in vivo.

In Vivo and In Vitro Characterization of a Plasmodium Liver Stage-Specific Promoter

Little is known about stage-specific gene regulation in Plasmodium parasites, in particular the liver stage of development. We have previously described in the Plasmodium berghei rodent model, a liver stage-specific (lisp2) gene promoter region, in vitro. Using a dual luminescence system, we now confirm the stage specificity of this promoter region also in vivo. Furthermore, by substitution and deletion analyses we have extended our in vitro characterization of important elements within the promoter region. Importantly, the dual luminescence system allows analyzing promoter constructs avoiding mouse-consuming cloning procedures of transgenic parasites. This makes extensive mutation and deletion studies a reasonable approach also in the malaria mouse model. Stage-specific expression constructs and parasite lines are extremely valuable tools for research on Plasmodium liver stage biology. Such reporter lines offer a promising opportunity for assessment of liver stage drugs, characterization of genetically attenuated parasites and liver stage-specific vaccines both in vivo and in vitro, and may be key for the generation of inducible systems.

Transcriptional silencing and activation of paternal DNA during Plasmodium berghei zygotic development and transformation to oocyst.

SummaryThe malaria parasite develops sexually in the mosquito midgut upon entry with the ingested blood meal before it can invade the midgut epithelium and embark on sporogony. Recent data have identified a number of distinct transcriptional programmes operating during this critical phase of the parasite life cycle. We aimed at characterizing the parental contribution to these transcriptional programmes and establish the genetic framework that would guide further studies of P lasmodium zygotic development and ookinete‐to‐oocyst transition. To achieve this we used in vitro and in vivo cross‐fertilization experiments of various parasite lines expressing fluorescent reporters under the control of constitutive and stage‐specific promoters. The results revealed that the zygote/ookinete stage exhibits a maternal phenotype with respect to constitutively expressed reporters, which is derived from either maternal mRNA inheritance or transcription of the maternal allele. The respective paternal alleles are silenced in the zygote/ookinete but reactivated after midgut invasion and transformation to oocyst. Transcripts specifically produced in the zygote/ookinete are synthesized de novo by both parental alleles. These findings highlight a putative role of epigenetic regulation of P lasmodium zygotic development and add substantially to the emerging picture of the molecular mechanisms regulating this important stage of malaria transmission.

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins.

We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.