A mutually-exclusive splicing poly-transgene expression system and method of use thereof

Abstract

A method of producing a plurality of proteins; it includes providing a transgenic organism with cells that have been modified to yield an alternatively spliced cassette section that has transgenes of interest at locations of spliced alternates that are separated by intronic sequences; and purifying proteins of interest expressed by the transgenes of interest in the transgenic organism following mutually exclusive splicing, thereby resulting in the plurality of proteins composed of the purified proteins of interest, and wherein the transgenic organism is an arthropod.

Description

A MUTUALLY -EXCLUSIVE SPLICING POLY-TRANSGENE EXPRESSION SYSTEM AND METHOD OF USE THEREOF

[0001] The present disclosure claims priority from U.S. provisional patent application No. 63 / 702,951 filed on October 3, 2024, incorporated herein by reference.Technical Field

[0002] The present disclosure relates to gene expression, and more particularly to systems for expressing a plurality of transgenes.Background

[0003] Proteins evolve within complexes of functional units, and in structural biology the detection of a protein’s structure within its multiprotein complex is important for understanding its biological function and evolutionary relationship (Ban et al., 2000; Bhattacharya, 2009; Stahl et al., 2024; Wiederstein et al., 2014). In cellular and systems biology, multiple molecules often need to be simultaneously manipulated to examine their roles and interactions within a cell or organism. In bacteria and yeast, multiple genes can be expressed using a promoter for each gene, but repeats of the same promoter can result in homologous recombination (Agmon et al., 2009). In multi-cellular organisms such as animals, it can be challenging to express multiple transgenes.

[0004] Multiple genes can be expressed using a single polycistronic mRNA strand (Kozak, 1999). For example, an internal ribosome entry site (IRES) is used by the poliovirus to initiate translation (Pelletier and Sonenberg, 1988), but can be used for bi-cistronic expression of two genes. An IRES sequence can be placed between an upstream gene and a downstream gene so that a ribosome can bind not only to the 5’ cap of the mRNA to translate the upstream gene, but also the IRES site to translate the downstream gene. However, the gene downstream of the IRES site is often translated at a much lower rate than the upstream gene and only two genes at a time is possible (Mizuguchi et al., 2000). Polycistronic expression can also be achieved using RNA sequences that encode cis-acting hydrolase element (CHYSEL) peptides (Doronina et al., 2008). CHYSEL polypeptides are commonly known as “2A” and “2A-like” peptides, and are used by RNA viruses to express each of its viral genes in a single mRNA strand. Synthesis of the CHYSEL polypeptide causes steric hindrance at the exit tunnel of the ribosome, causing the ribosome to skip the last bond of the CHYSEL peptide chain and restart on the next peptide (Donnelly et al., 1997; Martin D. Ryan GL, 2002; Ryan et al., 1991). Genes placed upstream and downstream of the CHYSEL peptide sequence can be co-translationally co-expressed at an almost stoichiometric ratio(Lo et al., 2015). Up to four genes can be simultaneously expressed (quad-cistronic) using this technique, but at decreasing efficiency for each downstream gene (Liu et al., 2017). Finally, the MultiLabel technique in mammalian cells can express up to five genes (Kriz et al., 2010), and is based on the MultiBAC baculovirus expression system used in moth Spodoptera frugiperda cell lines (Bieniossek et al., 2012; Sari et al., 2016).Summary

[0005] Alternative splicing is nature’s way of producing molecular diversity from a single gene. An extreme example of alternative splicing is the Drosophila Down syndrome cell adhesion moleculel (Dscaml) gene. Dscam is a cell surface receptor that is involved in several aspects of neural development, neural plasticity, and immune recognition (Schmucker and Chen, 2009). The Dscaml gene can produce tens of thousands of different isoforms through mutually exclusive splicing. It has 20 constitutive exons and 4 variable exon clusters: 4, 6, 9, and 17 which can be alternatively spliced in a mutually exclusive manner within each cluster (Schmucker et al., 2000) (Figure 1A). Within each of these variable exon clusters, the cell will select one and only one of the possible alternatives to splice into the mRNA. In variable exon 4, there will be only 1 out of 12 alternates spliced, in variable exon 6 only 1 out of 48 will be chosen, in exon 9 only 1 out of 33 will be chosen, and in exon 17 one or the other alternate is chosen, all incorporated into a single mRNA strand (as illustrated in Figure 1A). This splicing occurs for every round of Dscaml transcription, so there will be many mRNA molecules that each contain a different exon variant at each of the variable exon clusters.

[0006] Here, it is shown that placing the entire Dscam exon 4 alternatively spliced cassette section into a DNA plasmid under UAS control, and replacing each of the twelve exon 4 variants with a gene, allows each gene to be expressed simultaneously in a cell. Cell- or tissue-specific expression is controlled using the Gal4 / UAS system (Brand and Perrimon, 1993). It is also shown that alternative splicing of polycistronic dsRNA can induce RNAi against multiple genes. The technology is validated by rewiring Drosophila sensory neurons axons in vivo by simultaneously expressing several large cell surface receptors solely within the neuron, using the poly-transgene expression system (PXGS) described herein.

[0007] The present disclosure relates to mutually-exclusive poly transgene expression systems and methods of use thereof. The mutually-exclusive poly transgene expression system is derived from an alternatively spliced cassette section originating from an arthropod, where the alternativelyspliced cassette section is configured to perform, during transcription, mutually exclusive splicing of endogenous alternate genes located in the alternatively spliced cassette section separated by intronic sequences. The endogenous alternate genes are replaced by transgenes of interest, where these transgenes of interest undergo mutually exclusive splicing when transcribed within the transgenic organism. The transgenic organism thereby produces a plurality of proteins expressed by the transgenes of interest following the mutually exclusive splicing occurring during transcription of the alternatively spliced cassette section containing the transgenes of interest.

[0008] A broad aspect is a method of producing a plurality of proteins. The method includes providing a transgenic organism with cells that have been modified to yield an alternatively spliced cassette section that has transgenes of interest at locations of spliced alternates that are separated by intronic sequences; and purifying proteins of interest expressed by the transgenes of interest in the transgenic organism following mutually exclusive splicing, thereby resulting in the plurality of proteins composed of the purified proteins of interest, and wherein the transgenic organism is an arthropod.

[0009] In some embodiments, a genetic sequence upstream of the alternatively splicing cassette section may include more than one start codon that have been modified to insert a mutation in at least one of the more than one start codons.

[0010] In some embodiments, each of the more than one start codon may have been modified to insert a mutation, thereby resulting in a single functional start codon.

[0011] In some embodiments, the sequence downstream of the alternate cassettes may have been modified to insert a tag, thereby resulting in every spliced alternate having the tag associated thereto

[0012] In some embodiments, the arthropod may be an insect.

[0013] In some embodiments, the insect may be Drosophila melanogaster, and wherein the alternatively spliced cassette section may be derived from a Drosophila Down syndrome cell adhesion molecule 1 gene.

[0014] In some embodiments, the alternatively spliced cassette section may contain a derivation of Exon 4 of the Drosophila Down syndrome cell adhesion moleculel gene.

[0015] In some embodiments, the arthropod may be a crustacean.

[0016] In some embodiments, the plurality of protein may be used for vaccine production.

[0017] In some embodiments, the alternatively spliced cassette section may have been furthermodified to remove or duplicate one or more of the endogenous alternates to increase or decrease a number transgene of interest substitutions that replace the endogenous alternates in the alternatively spliced cassette section.

[0018] Another broad aspect is a plurality of proteins that has been produced by performing the method as described herein.

[0019] Another broad aspect is a mutually-exclusive poly transgene expression system including: an upstream nucleotide sequence; a downstream nucleotide sequence; and an alternatively spliced cassette section located between the upstream nucleotide sequence and the downstream nucleotide sequence, wherein the alternatively spliced cassette section comprises a plurality of transgenes of interest, wherein each transgene of interest of the plurality of transgenes of interest is substituting one of a plurality of endogenous alternates; and intron sequences located between the transgenes of interest of the plurality of transgenes of interest.

[0020] In some embodiments, the alternatively spliced cassette section may be derived from a Drosophila Down syndrome cell adhesion molecule 1 gene.

[0021] In some embodiments, the alternatively spliced cassette section may be derived from Exon 4 of the Drosophila Down syndrome cell adhesion molecule 1 gene.

[0022] In some embodiments, the upstream genetic sequence may have been modified to include at least one mutation in each start codon except for a first start codon present in a native genetic sequence from which the upstream genetic sequence has been derived.

[0023] Another broad aspect is a cell that has been transfected with the mutually-exclusive poly transgene expression system as described herein.

[0024] Another broad aspect is a transgenic arthropod that is constituted at least in part from the cells described herein.

[0025] Another broad aspect is a method of generating a mutually-exclusive poly transgene expression system, comprising replacing endogenous alternates of an alternatively spliced cassette section with transgenes of interest, wherein each of the endogenous alternates is replaced by one of the transgenes of interest.

[0026] In some embodiments, the endogenous alternates may include all of the endogenous alternates of the alternatively spliced cassette section.

[0027] In some embodiments, the method may include identifying start codons in a genetic sequence upstream from the alternatively spliced cassette; and inserting point mutations in a subsetof the start codons.

[0028] In some embodiments, the method may include removing or duplicating one or more of the endogenous alternates in order to decrease or increase a number of transgenes of interest that can be accommodated by the alternatively spliced cassette section.

[0029] In some embodiments, the alternatively spliced cassette section may be that of a Drosophila Down syndrome cell adhesion molecule 1 gene.

[0030] In some embodiments, the alternatively spliced cassette section may be Exon 4 of the Drosophila Down syndrome cell adhesion molecule 1 gene.Brief Description of the Drawings

[0031] The invention will be better understood by way of the following detailed description of embodiments of the invention with reference to the appended drawings, in which:

[0032] Figure 1A is a diagram illustrating the present system taking advantage of the mutually exclusive splicing property of Dscam. Endogenous Dscaml mRNA is mutually exclusively spliced in four exons during transcription, exons 4, 6, 9, and 17. For each of the mutually exclusively spliced exons, with every round of transcription, one and only one of the possible exon variants is in the final mRNA product and ultimately the protein product.

[0033] Figure IB is a diagram illustrating the present system taking advantage of the mutually exclusive splicing property of Dscam. The entire Dscam exon 4 cluster is inserted downstream of a UAS sequence to create the PXGS vector. A 6* histidine tag is added at the end of exon 5 to distinguish PXGS expression from endogenous Dscam exon 4 expression. Any gene of interest (GOI) can then be inserted to replace any of the 12 exon 4 alternates for expression. With each round of transcription, only a single transgene is selected for incorporation into the final mRNA product.

[0034] Figure 1C is a diagram illustrating the present system taking advantage of the mutually exclusive splicing property of Dscam. Exon 4 mutually exclusive splicing is maintained in PXGS. PXGS vectors with and without a 6* histidine tag were co-transfected with Actin5c-Gal4 in Drosophila S2 cells. As a positive control, reverse transcription (RT) of Exon 4 was performed on untransfected S2 cells to identify the endogenous Dscam Exon 4 mRNA. RT using PXGS-specific reverse primers on S2 cells was performed, with the no Actin5c-Gal4 co-transfection (PXGS only) as negative controls. PCR of Exon 4 in the PXGS vectors with and without a 6><His-tag showed DNA bands at the expected 500 bp to 600 bp sizes.

[0035] Figure 2 includes a diagram and images illustrating that the present system can express multiple fluorophores simultaneously in vivo. Crossing the pan-neuronal nSyb-Gal4 driver line with the transgenic fly UAS-PXGS_GFP4.1-BFPnols4.2-iRFP4.11-RFP4.12 demonstrates that nearly all neurons can splice and express multiple transgenes in vivo. However, the fluorescence intensity for any single color was low as its production was divided among 12 Exon 4 alternates.

[0036] Figure 3 includes a diagram and images illustrating that the present system can drive expression in non-neural cells. Crossing the transgenic fly UAS-PXGS_iRFPnols4.1- COX8::RFP4.2-BFPnols4.3-mCD8::GFP4.4-iRFPnols4.5-COX8::RFP4.6-mCD8::GFP4.8- BFPnols4.9-iRFPnols4.10-BFPnols4.12 to the glial Repo-Gal4 driver (left column) or to the ubiquitous Tubulin-Gal4 driver (right column) showed expression in non-neural cells. The subcellular localization of the fluorophores was observed in the flight muscle (right column), but was less apparent in glial cells in the brain (left column). Scale bars are 50pm.

[0037] Figure 4A includes diagrams and an image illustrating that the present system can express multiple cell surface receptors in vivo to manipulate neural wiring. A representative example of the pSc axonal branching pattern in 455-Gal4; PXGS fluorophores controls is given. A schematic of the three PXGS constructs expressing different combinations of cell surface receptors is shown on the right.

[0038] Figure 4B are images showing two representative examples of the pSc axonal branching in 455-Gal4 / +; PXGS_dprl24.1-dpr84.2-Gal44.3 / + flies (left images), 455-Gal4 / +; PXGS_kekl4.7-kirre4.8-tutl4.9 / + flies (middle images), and 455-Gal4 / +; PXGS Toll-64.10- Bsg4.1 l-sli4.12 / + flies (right images). Yellow arrows point to the pSc axon exiting the thoracic ganglion anteriorly. White arrowheads denote missing branches.

[0039] Figures 4C and 4D are graphs illustrating overexpression of multiple cell surface receptors significantly increases axonal targeting errors. A frequency distribution shows the five error types found in the PXGS-cell surface receptor flies. A schematic for each error is shown below the x-axis, where blue branches indicate ectopic branches. Posterior shortening and contralateral anterior missing branches occurred significantly more frequently in 455-Gal4 / +; PXGS_dprl24.1-dpr84.2-Gal44.3 / + flies and 455-Gal4 / +; PXGS_Toll-64.10-Bsg4.11-sli4.12 / + flies. Both 455-Gal4 / +; PXGS_dprl24.1-dpr84.2-Gal44.3 / + flies and 455-Gal4 / +; PXGS Toll- 64.10-Bsg4.11-sli4.12 / + flies had significantly reduced total branch length and branch number compared to the control (left graphs). The 455-Gal4 / PXGS_fluorophores andPXGS _fluorophores / + both had significantly reduced total branch length compared to 455-Gal4 / +, most likely due to genetic background. 455-Gal4 / PXGS fluorophores and PXGS _fluorophores / + flies had no significant differences in branch length (right graphs). Dotted lines mark the midline. Scale bars are 50pm. Statistical significance comparisons to control are indicated directly above the bar representing each genotype. No asterisks indicate the comparison was not significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0040] Figure 5A includes images illustrating a complete replacement of an Exon 4 alternate can properly splice and produce functional protein. Replacing Exon 4.1 with the gene for GFP produces green fluorescence when co-transfected with Actin5C-Gal4 in S2 cells. Blue (upper left), green (upper right), red (lower left), and far red (lower right) channels are shown. Scale bars are 5 pm.

[0041] Figure 5B is a diagram and a graph illustrating a complete replacement of an Exon 4 alternate can properly splice and produce functional protein. PXGS-specific RT-PCR of different exon 4 variants in transfected S2 cells verified their appropriate splicing and expression. GFP mRNA band is expected at 716 bp, the remaining possible exon 4 variants are expected at -500 bp (denoted with a *), Exon 4.2 is expected at 406 bp, and Exon 4.1 is not expected.

[0042] Figure 6A is a diagram and images illustrating that replacing 10 of the 12 Exon 4 alternates with fluorescent protein transgenes increases the expression pattern in neurons. Cotransfection of UAS-PXGS_iRFPnols4.1-COX8::RFP4.2-BFPnols4.3-mCD8::GFP4.4- iRFPnols4.5-COX8::RFP4.6-mCD8::GFP4.8-BFPnols4.9-iRFPnols4.10-BFPnols4.12 fly with Actin5C-Gal4 in S2 cells produced mRNA at the expected sizes (iRFP = 1044bp, BFP = 810bp, RFP = 804bp, GFP = 270bp), and produced fluorescent cells. Scale bars are 10pm.

[0043] Figure 6B is a diagram and images illustrating that replacing 10 of the 12 Exon 4 alternates with fluorescent protein transgenes increases the expression pattern in neurons. Crossing the pan-neuronal nSyb-Gal4 driver line with the transgenic fly UAS-PXGS_iRFPnols4.1- COX8::RFP4.2-BFPnols4.3-mCD8::GFP4.4-iRFPnols4.5-COX8::RFP4.6-mCD8::GFP4.8- BFPnols4.9-iRFPnols4.10-BFPnols4.12 resulted in all neurons expressing all four fluorophores at higher levels than the four fluorophore PXGS fly. The differential subcellular localizations of the fluorophores can be observed at higher magnifications in the optic lobe. White arrowheads in the zoomed in merged image (bottom right) point to cells that express all four fluorophores evenly. Scale bars are 50pm, 50pm, and 5pm respectively from left to right.

[0044] Figure 7 is an image illustrating that the present system can express multiple cell surface receptors in S2 cells. UAS-PXGS_dpr84.1-dprl24.2-Gal44.3, UAS-PXGS_kekl4.7- kirre4.8-tutl4.9, and UAS-PXGS_ Toll-64.10-Bsg4.11_sli4.12 were each co-transfected into S2 cells with Actin5C-Gal4. RT-PCR on each gene was performed 48 hours after transfection. The PCR products for UAS-PXGS_dpr84.1-dprl24.2-Gal44.3 are shown in the left side, UAS- PXGS_kekl4.7-kirre4.8-tutl4.9 in the middle, and UAS-PXGS_ Toll-64.10-Bsg4.11_sli4.12 on the right. The expected sizes for each gene’s PCR product is shown. Gal4 mRNA from PXGS expression was not detected likely due to a mutation within the upstream intronic sequence that may interfere with its splicing.

[0045] Figure 8 includes a diagram and images illustrating that the present system can express dsRNA for RNAi knockdown. Flies expressing dsRNA against RFP did not have red fluorescence, but still expressed BFP. The low iRFP signal is possible due to the off-target effects of the RNAi. Scale bar is 50pm.

[0046] Figure 9 is an image illustrating that the present system can express 13 large genes to re-create synthetic biology pathways. Actin-Gal4 flies were crossed to PXGS flies expressing 13 genes to synthesize the carbon fixing enzyme, Ribulose- 1,5 -bisphosphate carboxylase / oxygenase (RuBisCO). RT-PCR verified that all 13 genes were expressed in the flies.Detailed Description

[0047] The mutually-exclusive poly transgene expression systems of the present disclosure are derived from an alternatively spliced cassette section of an arthropod, where the endogenous alternate genes are replaced by transgenes of interest. When transcription of the alternatively spliced cassette section occurs, mutually exclusive splicing is performed, thereby resulting in a selection of one of the transgenes of interest during a transcription session, where a plurality of transcription sessions results in a mixture of RNA transcribed from different transgenes of interest selected via the mutually exclusive splicing. The organism thereby yields a plurality of different proteins of interest resulting from the transgenes of interest.

[0048] Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is as “including, but not limited to.”

[0049] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with theembodiment is included in at least one embodiment. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0050] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and / or” unless the content clearly dictates otherwise.

[0051] From the foregoing it will be appreciated that, although specific embodiments have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the teachings. Accordingly, the claims are not limited by the disclosed embodiments.

[0052] DEFINITIONS:

[0053] By “alternatively spliced cassette section”, it is meant an exon, or a part of an exon, that has a sequence suitable for permitting mutually exclusive splicing during transcription in an arthropod or arthropod-derived cell. The alternatively spliced cassette section includes alternate genes or multiple transgenes of interest that are separated by intronic sequences, that are selected during transcription by the mutually exclusive splicing.

[0054] By “intronic sequence”, it is meant sequences that are spliced out or not included in the final mRNA product following transcription.

[0055] The term “nucleic acid sequence”, as used herein, as well as “nucleotide sequence”, “polynucleotide sequence”, may be used interchangeably herein and refer to a contiguous nucleic acid sequence. The sequence can be either single stranded or double stranded DNA or RNA, such as an mRNA.

[0056] The term “nucleic acid”, as used herein, refers to any compound that includes a polymer of nucleotides, and may be referred to as a polynucleotide. Exemplary nucleic acids include, but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), threose nucleic acids (TNA), glycol nucleic acids (GNA), peptide nucleic acids (PNA), locked nucleic acids (LNA), etc. The term includes triple-, double- and single- stranded deoxyribonucleic acids (DNA), and triple- , double- and single-stranded ribonucleic acid (RNA). The term also includes optionally a modification by, for instance, capping, alkylation, etc.

[0057] The term “pharmaceutical composition”, as used herein, refers to a compound in accordance with the present disclosure, or a pharmaceutically acceptable salt thereof, in a form suitable for, e.g., oral administration (e.g. through inhalation) or parenteral administration.

[0058] The term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient”, as used herein, refers to a substance useful in the preparation of a pharmaceutical composition and includes, for instance, one or more of diluents, surfactants, preservatives, buffering agents, isotonic agents, salts, excipients, lubricants, wetting agents, flavoring, etc. Exemplary excipients include, but are not limited to, calcium carbonate, calcium phosphate, citric acid, hydroxyl methyl cellulose, gelatin, maltitol, providone, palmitate, sorbitol, starch, stearic acid, vitamins, etc.

[0059] The term “pharmaceutically acceptable salts”, as used herein, refers to derivatives of the disclosed compounds by converting an acid or base moiety to its salt form (e.g. by reacting a base group with a suitable organic acid).

[0060] The term “prevent”, “preventing” or “prevention”, as used herein, refers to the prophylactic treatment of a disease or disorder, or delaying the onset or progression of a disease or disorder.

[0061] In the present disclosure, by “subject”, it is meant a mammal, such as a human. The term “subject” should not bring on any limitations as to the sex or age.

[0062] The term “treat”, “treating” or “treatment”, as used herein, refers to alleviating or improving the outcome of the subject with regard to a given disease or disorder, which may be quantifiable by improving at least one physical parameter, ailment or biomarker of the subj ect with the disease or disorder.

[0063] The following exemplary studies are provided to enable the skilled person to better understand the present disclosure. As they are but illustrative and representative examples, they should not limit the scope of the present disclosure. They are only added for illustrative and representative purposes. It will be understood that other exemplary studies may be used to further illustrate and represent the present disclosure without departing from the present teachings.

[0064] EXEMPLARY STUDY 1: Inserting the Poly-transgene Expression System (PXGS) into plasmids

[0065] In the present disclosure, Dscaml is also referred to herein as Dscam. It will be understood that even though the present examples relate to Dscaml, the teachings presented hereinmay also be applied to other gene paralogs that have hypervariable mutually exclusive alternative splicing, including one of the four Drosophila melanogaster Dscam gene paralogs that has the hypervariable mutually exclusive alternative splicing. Moreover, other gene homologs that exhibit hypervariable mutually exclusive alternative splicing may be found in other insects and crustaceans.

[0066] The entire Dscam exon 4 region was amplified along with 300 basepairs (bp) at the 3’ end of Dscam exon 3 and 30 bp at the 5’ start of exon 5 from Drosophila melanogaster genomic DNA, totaling 6.7 kilobasepairs. The PCR was carried out for 1 min at 94°C, 1 min at 55°C, and 15 min at 72°C for 35 cycles using CloneAmp (Takara Bio Inc. USA, Mountain View, CA) (Celotto and Graveley, 2001; Haussmann et al., 2019; Kreahling and Graveley, 2005). There are several ATG codons (coding for Methionine) within exon 3, which would then create several unintended translation start sites, adding up to 70 unwanted, in-frame amino acids attached to the N-terminus of every PXGS transgene. To eliminate this problem, three point-mutations were inserted into exon 3 (Table 1).

[0067] The Dscam fragment was then inserted into the pJFRC7-20XUAS-IVS-mCD8::GFP expression vector (Pfeiffer et al., 2010) using Gibson assembly (Gibson et al., 2009). The fragment was inserted downstream of the UAS sequences and replaced the mCD8::GFP sequence. A 6><Histidine tag was added at the end of exon 5 to distinguish PXGS sequences in reverse transcriptase PCR. Gibson assembly and transformation were performed with the In-Fusion® HD Cloning Kit and Stellar™ Competent Cells following the kit instructions (Takara Bio Inc. USA, Mountain View, CA).

[0068] EXEMPLARY STUDY 2: INSERTING TRANSGENES INTO PXGS

[0069] Transgene insertions into the exon 4 alternates within our different PXGS plasmids were created using the Molecular Assembly feature in GeneDig.org (Suciu et al., 2015), primarily using gene synthesis, Gibson assembly, and restriction enzyme digest and ligation. Exon 4 variants were replaced with fluorophore genes codon optimized for Drosophila, or cell surface molecule genes, or hairpin RNA sequences.

[0070] The GFP gene was based on the mNeonGreen gene (Ceolin et al., 2020). The BFP and RFP genes were obtained from a plasmid previously generated in our lab (Lo and Chen, 2019). The iRFP gene was based on miRFP670 (Shcherbakova et al., 2016) and codon optimized for Drosophila and synthesized by Integrated DNA Technology (Coralville, IA). To sequester thefluorophores into subcellular compartments to help distinguish their expression, transmembrane CD8 (mCD8) was used for membrane localization, a nucleolar localization signal (nols) for nuclear labelling, and C0X8 for mitochondrial localization.

[0071] Sequences for the eight cell surface receptors, Bsg (798 bp), dpr8 (891 bp), dprl2 (1,035 bp), kekl (2,643 bp), kirre (2,871 bp), sli (4,443 bp), Toll-6 (4,545 bp), and tutl (2,892 bp) were obtained from RT-PCR on wildtype flies using oligo d(T) reverse primer to generate the cDNA, and gene-specific forward and reverse primer to amplify the coding sequences. All DNA constructs were verified by colony PCR and Sanger sequencing.

[0072] EXEMPLARY STUDY 3: CELL CULTURE

[0073] All PXGS transgenes were first verified using RT-PCR after transfection into S2 cells. Drosophila S2 cells were cultured at 25°C in Ex-Cell 420 Medium (Sigma-Aldrich, St. Louis, MO). Cells were transfected with 5 pg of plasmid DNA in 35 mm dishes using Lipofectamine (ThermoFisher, Waltham). 48 hours after transfection, RNA was extracted from the S2 cells and reverse transcription was performed on transfected and untransfected cells. PXGS-specific reverse primers for the 6><His tag or for the overlap between exon 5 and the vector were used to identify PXGS transgenes, and oligo-d(T) primers were used for endogenous Dscam mRNA alone. Endogenous Dscam exon 4 was used as the positive controls, and PXGS plasmid with no Actin5C- Gal4 transfection was used as the negative controls. PCR was performed using gene-specific forward and reverse primers.

[0074] EXEMPLARY STUDY 4: FLY STOCKS

[0075] To reduce variability due to environmental conditions and genetic background, flies were reared at 25°C on standard cornmeal under 12h light / 12h dark cycles. To express PXGS fluorophores in different cell types, the pan-neuronal nSyb-Gal4, glial Repo-Gal4, ubiquitous Tubulin-Gal4, and scutellar 455-Gal4 drivers were used. PXGS plasmids that were verified for expression in S2 cells were used to make transgenic animals. Transgenic Drosophila lines were generated using PhiC31 integrase-mediated transgenesis performed by BestGene, Inc (Chino Hills, CA).

[0076] EXEMPLARY STUDY 5: CARBOCYANINE DYE LABELING AND DROSOPHILA IMAGING SAMPLE PREPARATION

[0077] Drosophila mechanosensory axonal arbors were labelled using carbocyanine dye injection onto the pSc neurons, as previously described (Chen et al., 2006; Cvetkovska et al., 2013;Dos Santos et al., 2019; Kays et al., 2014; Neufeld et al., 2011). The left and right pSc bristles of 2-day old female flies were plucked and the thorax was left overnight in fixative solution (3.7% paraformaldehyde in 0.2 M carbonate-bicarbonate buffer, pH 9.5). The left and right pSc neurons were labelled using the carbocyanine tracers, Dil or DiD (ThermoFisher). The flies were left undisturbed and partially immersed in 0.2 M carbonate-bicarbonate buffer at pH 9.5. After 48 hours, the thoracic ganglion was dissected and imaged.

[0078] EXEMPLARY STUDY 6: IMAGE ACQUISITION

[0079] Fluorescent images were acquired using an AxioScope Al epifluorescence microscope (Carl Zeiss) with a 40* objective (N.A. 1.0) or an FV1000 laser-scanning confocal microscope (Olympus) with a 40* objective (N.A. 1.3). For mechanosensory axon imaging, 10- 20 images were taken at different focal planes. Maximal intensity z-proj ections were stacked to generate the final image. Images were adjusted only for contrast and brightness when required. Transmitted light images were used to evaluate dissection and dye labeling quality and to measure thoracic ganglia width.

[0080] EXEMPLARY STUDY 7: IMAGE ANALYSIS

[0081] Only images without central nervous system damage or surface occlusions were included for data analysis. Measurements of axonal branch length and branch number were performed blind to genotype by randomly distributing the axonal arbor data of the total dataset. Custom-written software in MATLAB was used to perform the quantitative image analysis.

[0082] To determine the baseline variability in wildtype pSc mechanosensory neurons, the total number of branches and branch lengths per arbor were measured from thirty control 455- Gal4 / + animals. A skeleton of the control 455-Gal4 / + axonal arbor was then created based on the frequency of occurrence of each branch, which was found to be consistent with our previous measurements (Chen et al., 2006; Cvetkovska et al., 2013; Dos Santos et al., 2019; Kays et al., 2014; Neufeld et al., 2011). an ectopic branch is defined as any branch that has less than 30% occurrence in the wildtype. Representative examples of axonal arbors in figures were randomly chosen from the sets of all axonal arbor images of the genotype.

[0083] EXEMPLARY STUDY 8: STATISTICAL ANALYSIS

[0084] Statistical significance for total axonal branch length and number of branches was determined using one-way ANOVA followed by Dunnett’s multiple comparisons test. Statistical significance for frequency of occurrence was determined using a two-tailed t-test for proportions.Statistical analysis was performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, San Diego, California USA) and SPSS (version 25). The datasets, analyses, and materials used in the current study are available from the corresponding author upon request.

[0085] EXEMPLARY STUDY 9: REVIEWING THE RESULTS

[0086] Dscam mutually exclusive splicing is conserved in the PXGS system

[0087] Dscam variable exon 4 was verified to be properly spliced within an exogenous DNA construct. The last 300 bases of Dscam exon 3 was extracted through variable exon 4 to the first 30 bases of exon 5 from Drosophila melanogaster genomic DNA. A 6* Histidine DNA sequence was added to the end of the exon 5 sequence as a genetic tag. Dscam alternative splicing was also tested to see if the Dscam alternative splicing would be preserved after transcription under a UAS promoter (Figure IB), to allow it to be expressed conditionally using a Gal4 driver (Brand and Perrimon, 1993). The 6.7 kilobasepair Dscam exon 4 fragment was inserted into the expression vector pJFRC7-20XUAS, and co-transfected it along with Actin5C-Gal4 into Drosophila melanogaster S2 cells (Figure 1C). To determine whether the endogenous exon 4 alternates were properly spliced, reverse transcriptase PCR (RT-PCR) was performed using the 6* Histidine genetic tag to distinguish the UAS-transcripts from the endogenous Dscam mRNA transcripts. It was found that in S2 cells transfected with UAS-DscamExon4, the exogenous Exon 4 variants were produced at the expected sizes as the endogenous Exon 4 alternates (Figure 1C).

[0088] Inserting fluorescent protein genes in the endogenous exon 4 alternates

[0089] The mutually exclusive splicing of Dscam exon 4 is determined by its intronic structures (Hong et al., 2021; Kreahling and Gravel ey, 2005; Xu et al., 2019; Yue et al., 2016), but it is not certain that the exonic 4 sequences do not contribute. First, the green fluorescent protein (GFP) transgene was inserted inside of (i.e., surrounded by) the endogenous exon 4 alternates, which would result in the endogenous sequences being treated as untranslated regions (UTRs) of mRNA flanking the transgene, systematic narrowing down of the minimum endogenous Dscam exonic 4 sequences required for proper splicing was performed. In parallel, we tried to completely replace the entire exon 4.1 alternate with the GFP gene. To our surprise the GFP mRNA and functional fluorescence was expressed in S2 cells (Figures 5A-5B). Thus, only the intronic sequences within variable exon 4 are required for proper mutually exclusive splicing.

[0090] Replacing exon 4 variants with fluorophores

[0091] Verification was then performed that other exon 4 variants could be completelyreplaced to express any gene of interest within our UAS-DscamExon4 construct. Alternate exons 4.1, 4.2, 4.11, and 4.12 were replaced with GFP, blue fluorescent protein with a nucleolar localization signal (BFPnois), near-infrared fluorescent protein (iRFP), and red fluorescent protein (RFP) transgenes, respectively. After verification of their expression in S2 cells using RT-PCR, we generated a transgenic fly containing the UAS-DscamExon4 construct, UAS- DscamExon4_GFP4 l-BFPnols4 2-iRFP4"-RFP4 12. This fly was crossed to the pan-neuronal Gal4 driver nSyb-Gal4, and found that all four fluorophores were weakly to moderately expressed but not in all neurons (Figure 2). If Exon 4 alternative splicing is random, then after multiple rounds of transcription eventually all colors should be expressed. If Exon 4 splicing is deterministic, then some neurons may never express the splicing factors that select the missing colors. In other words, some neurons may not express specific splicing factors to allow for expression of specific Exon 4 alternates. An important caveat for this UAS-DscamExon4 construct is that for each round of transcription driven by the nSyb-Gal4, a neuron may splice any of the eight Exon 4 endogenous alternates (i.e., producing non-fluorescence) or any of the four fluorophores, so that the “signal” is diluted and divided, respectively, compared to if nSyb-Gal4 were driving expression of a standard UAS-fluorescent protein construct.

[0092] Regardless of if Dscam Exon 4 is random or deterministic, it was shown that Exon 4 alternates can be replaced by a gene of interest for poly-transgene expression and can be conditionally controlled by Gal4 expression. After transcription and splicing, any extraneous DNA surrounding the transgene such as Exon 3, Exon 5, and the 6* Histidine tag are untranslated mRNA regions (UTRs) and are not synthesized by the ribosome, as each transgene contains their own start and stop codons.

[0093] PXGS can drive expression in non-neuronal cells

[0094] Dscam is expressed in all cells, based on gene expression atlases for Drosophila, albeit at varying levels (Corrales et al., 2022; Li et al., 2022). Dscam has traditionally been studied in the nervous and immune systems. To verify that our new poly-transgene expression system (PXGS) can be used in cells beyond neurons and immune cells, a multi-color PXGS reagent was generated to label any cell that expresses Dscam by replacing ten exon 4 alternates with fluorophores. GFP, BFP, RFP, and iRFP were differentially localized to the membrane (mCD8::GFP), the nucleus (nols), or the mitochondria (COX8::RFP), and generated the construct PXGS iRFPnois4kCOXS : :RFP42-BFPnois43-mCD8 : : GFP44-iRFPnois4 5-COX8 : :RFP46-mCD8::GFP4-8-BFPnois4'9-iRFPnois4 10-BFPnois4 12. The PXGS expression was verified in S2 cells by co-transfection with Actin5C-Gal4 (Figure 6A) and proceeded to generate transgenic flies. The UAS-PXGS_iRFPnois4 1-COX8::RFP4 2-BFPnois43-mCD8::GFP4 4-iRFPnois4 5-COX8::RFP46- mCD8::GFP4-8-BFPnois4'9-iRFPnois4 10-BFPnois4 12flies were crossed to the pan-neuronal nSyb-Gal4 and the brains were imaged to verify expression in vivo (Figure 6B). Neurons were strongly fluorescent with green at the cell membrane, intracellular punctate red mitochondria, and overlapping blue and infrared nuclei. However, the blue channel was often dominated by autofluorescence of the tracheal tubes.

[0095] Crossing the UAS-PXGS_iRFPnois4 1-COX8::RFP4 2-BFPnois43-mCD8::GFP44- iRFPnols4 5-COX8::RFP46-mCD8::GFP4 8-BFPnols49-iRFPnois4 10-BFPnois4 12flies to the pan-glial Repo-Gal4 driver revealed expression of all four fluorophores across the brain (Figure 3). These PXGS flies were crossed to Tubulin-Gal4 for ubiquitous expression across the animal. Fluorescence expression was highest in the brain, but fluorescence was also observed in muscle. Multi-nucleated muscle cells were labelled with blue and near-infrared fluorescence, green cell membrane, and red mitochondria puncta (Figure 3). Similar to previous studies, the mCD8::GFP expression was observed concentrated at the immediate area surrounding the nucleus (Ralston and Hall, 1989).

[0096] Functional expression of PXGS transgenes

[0097] The genes for fluorescent proteins are <1 kilobase in size. To demonstrate that PXGS can express large, functionally relevant genes, eight cell surface receptors to mis-express were selected in a mechanosensory neuron. The eight genes were Bsg, dpr8, dprl2, kekl, kirre, sli, Toll- 6, and tutl. Three PXGS constructs were constructed: UAS-PXGS_dpr84 1-dprl24'2-Gal44'3, UAS- PXGS_kekl4'7-kirre4'8-tutl4'9, and UAS-PXGS_ Toll-64'10-Bsg4 11_sli4'12and their mRNA expression were verified in S2 cells (Figure 7) before generating fly lines. The gene for Gal4 itself was inserted into the Exon 4.3 position to create a transcriptional positive feedback loop for continuous gene expression. The 455-Gal4 driver line was used to mis-express these cell surface receptors solely within the pSc mechanosensory neuron (Cvetkovska et al., 2013; Dos Santos et al., 2019; Kays et al., 2014; Neufeld et al., 2011). The pSc mechanosensory neuron has a stereotyped axonal targeting pattern within the central nervous system, which can be used as an assay to identify molecules involved in axonal growth and targeting. Dscam isoforms are required for proper neural wiring (Chen et al., 2006), so overexpression of the PXGS constructs mayinterfere with the alternative splicing of endogenous Dscam isoforms by competing for the same splice factors, in a “sponging” effect. To account for this, the 455-Gal4 driver was crossed to the UAS-PXGS_iRFPnois4 1-COX8::RFP4 2-BFPnois43-mCD8::GFP4 4-iRFPnois4 5-COX8::RFP46- mCD8::GFP4 8-BFPnois49-iRFPnois4 10-BFPnois4 12flies (herein referred to as PXGS fluorophores) as our control lines (Figure 4A).

[0098] Compared to the control flies (n = 13), mis-expression of dpr8 and dprl2 (n = 20) or Toll-6, Bsg, and sli (n = 15) within the pSc resulted in a significant increase in axonal targeting errors (p < 0.0001, t-test) (Figures 4B, 4C). The axons frequently failed to extend posteriorly or anteriorly on the contralateral side. In extreme cases, the axon would stall at the entry point (Figure 4B). This resulted in significantly shorter total branch lengths and total branch numbers in these two fly lines, compared to 455-Gal4 / PXGS-fluorophores control (p < 0.05, ANOVA) (Figure 4D). Mis-expression of kekl, kirre, and tutl simultaneously in the pSc neuron (n = 15) frequently resulted in the axon exiting the thoracic ganglion (Figures 4B, 4C).

[0099] The control 455-Gal4 / PXGS-fluorophores flies were also found to have significantly shorter total branch lengths of the pSc axon (p < 0.05, ANOVA) compared to 455-Gal4 / + controls (n = 30). However, this is likely due to genetic background differences, and not a “sponging” effect of PXGS on Dscam splicing factors, because the PXGS -fluor ophore / + flies (within no Gal4) (n = 15) also had significantly shorter pSc branch lengths than 455-Gal4 / + flies. The difference in axonal branch lengths between PXGS -fluor ophore / + flies and 455-Gal4 / PXGS- fluorophores flies was not significant (Figure 4D).

[0100] EXEMPLARY STUDY 10: DISCUSSION

[0101] The poly-transgene expression system was created to allow any Drosophila biologist to simultaneously express multiple genes under conditional control. Compared to other multigene expression strategies, PXGS offers two distinct advantages. First, it can express many more transgenes than what was previously achievable. Each PXGS construct can express 12 transgenes, so that instead of a standard triple-transgenic animal containing three transgenes, a PXGS animal would have 36 transgenes. Second, the molecular biology to create a PXGS is straightforward and easier to work with. For example, using 2 A peptide DNA sequences for more than two genes can be technically challenging due to the repetition of the 2A sequences, which can confound the primer design step, the plasmid assembly, and the sequence verification. This difficulty is eliminated in PXGS. The DNA sequences of the intronic regions spanning each of the 12 exonalternates are different enough that molecular assembly primers do not have multiple binding sites. The turnaround time to generate a PXGS construct is around 3-4 weeks.

[0102] Whether a specific cell expresses all 12 Exon 4 alternates for PXGS is dependent on three things. First, the expression level of the Gal4 driver which dictates how many rounds of PXGS transcription will occur. This can be supplemented by including the gene for Gal4 within one of the alternates. Second, if the cell expresses Dscam at all, then whether or not the cell expresses the appropriate Exon 4 splicing factors. If Dscam exon 4 splicing is deterministic, then cells that do not express the correct splice factors could be made to, again by expressing the correct splicing factors using PXGS itself. However, all of the splice factors and mechanisms involved in Exon 4 are currently not known. Additionally, mis-expressing Dscam splicing factors would interfere with the endogenous Dscam splicing function and will likely adversely affect the cell given that those Exon 4 alternates were deterministically not spliced. Third and similarly, if Dscam Exon 4 splicing is random, there still may be a lack of appropriate splicing factors despite a high transcription rate. A high transcription rate of PXGS may also cause one Exon 4 alternate to be repeatedly selected due to the constitutive recruitment of its splicing factor.

[0103] For each round of transcription, the probability that any given Exon 4 alternate is not spliced is 11 out of 12. The number of endogenous Dscam isoforms expressed within a single cell can be used as a minimum number of rounds of transcription that must have occurred, and this is approximately 10 - 1000 isoforms (Schmucker and Chen, 2009). For ten rounds of transcription, the probability that a specific Exon 4 alternate is not spliced is (11 / 12)10, or 42%. After 50 rounds of transcription, the probability that a specific exon 4 alternate is not spliced is (11 / 12)50, or 1%.

[0104] If Dscam Exon 4 splicing is random, then modifying the number of alternates in PXGS is less likely to negatively affect the cell. Removing variants in Exon 4 does not affect splicing (Dong et al., 2023), thus PXGS can be customized to exactly the number of variants needed by removing or duplicating the Exon 4 alternates, mimicking what has occurred through evolution (B rites et al., 2013; Lee et al., 2010).

[0105] For systems level biological investigations, loss-of-function experiments on multiple genes simultaneously may be required. RNAi is a common approach used for loss-of-function analyses (Qiao et al., 2018). UAS-based expression of dsRNA allows for RNAi-mediated knockdown in specific cells. A UAS-PXGS_dsRNA-RFP4 1-BFPnois4'2- Gal44'3-Shibirets46- iRFP4 n-RFP4 12fly was generated to knockdown the RFP expression in position 4.12, whileleaving the BFPnois in position 4.2 unaffected. Crossing this fly to nSyb-Gal4, RFP was found to not be expressed in the brain, but nuclear BFP was clearly visible (Figure 8). The strong BFP signal was likely due to the Gal4 in position 4.3 that created a positive feedback, but this may have also increased the off-target effects of the RNAi, as no iRFP was observed. These experiments demonstrate the potential to use PXGS for polycistronic gene knockdown. It is not clear which RNAs may be properly spliced and processed in PXGS, such as guideRNAs, circular RNA, complex secondary structures, or other non-coding RNAs.

[0106] The PXGS system will also allow Drosophila melanogaster to be a new model system synthetic biology, and for biosynthesis and biomanufacturing in S2 cells (Coker et al., 2022). For example, PXGS was used to express the 13 genes required for synthesis of the carbon fixing enzyme, Ribulose- 1,5 -bisphosphate carb oxy lase / oxygenase, RuBisCO (Aigner et al., 2017; Gleizer et al., 2019). The fly UAS-PXGS_rbcLl-PQR-rafl4 1-cpn60al42-cpn60bl4 3-rbCS3B44- cpn204'5-raf24'6-rbcx24'7-bsd24'8-RiBi-Is4'9- PRK4 10- PGLP14 11- G0X14 12was crossed with nSyb- Gal4 and it was verified that all 13 genes were transcribed (Figure 9). The two genes, rbcLl and rafl were inserted into position 4.1 with a CHYSEL Protein Quantitation Reporter (PQR) linker specific to Drosophila (Lo et al., 2015), demonstrating that a poly-cistronic RNA within the polycistronic PXGS can generate even larger numbers of transgenes. Thus, PXGS can be used to express complex enzymatic or molecular pathways can in Drosophila. The technology can be expanded to nearly all arthropods (B rites et al., 2013; Lee et al., 2010; Schmucker and Chen, 2009), where any insect or crustacean can be genetically modified to express multiple transgenes such as mosquitos, silkworm moth, honeybees, and aquaculture shrimp such as the king prawn.

[0107] Representative, non-limiting examples of the present invention were described above in detail with reference to the attached drawing. This detailed description is merely intended to teach a person of skill in the art further details for practicing preferred aspects of the present teachings and is not intended to limit the scope of the invention. Furthermore, each of the additional features and teachings disclosed above and below may be utilized separately or in conjunction with other features and teachings to provide useful lipid nanoparticle delivery systems and methods of using same within the subject.

[0108] Moreover, combinations of features and steps disclosed in the above detailed description, as well as in the experimental examples, may not be necessary to practice the invention in the broadest sense, and are instead taught merely to particularly describe representativeexamples of the invention. Furthermore, various features of the above-described representative examples, as well as the various independent and dependent claims below, may be combined in ways that are not specifically and explicitly enumerated in order to provide additional useful embodiments of the present teachings.

[0109] All features disclosed in the description and / or the claims are intended to be disclosed separately and independently from each other for the purpose of original written disclosure, as well as for the purpose of restricting the claimed subject matter, independent of the compositions of the features in the embodiments and / or the claims. In addition, all value ranges or indications of groups of entities are intended to disclose every possible intermediate value or intermediate entity for the purpose of original written disclosure, as well as for the purpose of restricting the claimed subject matter.

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Claims

What is claimed is:

1. A method of producing a plurality of proteins comprising: providing a transgenic organism with cells that have been modified to yield an alternatively spliced cassette section that has transgenes of interest at locations of spliced alternates that are separated by intronic sequences; and purifying proteins of interest expressed by the transgenes of interest in the transgenic organism following mutually exclusive splicing, thereby resulting in the plurality of proteins composed of the purified proteins of interest, and wherein the transgenic organism is an arthropod.

2. The method according to claim 1, wherein a genetic sequence upstream of the alternatively splicing cassette section includes more than one start codon that have been modified to insert a mutation in at least one of the more than one start codons.

3. The method according to claim 2, wherein each of the more than one start codon except for one start codon has been modified to insert a mutation, thereby resulting in a single upstream functional start codon.

4. The method according to claim 1, wherein the sequence downstream of the alternate cassettes has been modified to insert a tag, thereby resulting in every spliced alternate having the tag associated thereto.

5. The method according to any one of claims 1 to 4, wherein the insect is Drosophila melanogaster, and wherein the alternatively spliced cassette section is derived from a Drosophila Down syndrome cell adhesion molecule 1 gene.

6. The method according to claim 5, wherein the alternatively spliced cassette section contains a derivation of Exon 4 of the Drosophila Down syndrome cell adhesion molecule 1 gene.

7. The method according to any one of claims 1 to 6, wherein the plurality of protein is used for vaccine production.

8. The method according to any one of claims 1 to 7, wherein the alternatively spliced cassette section has been further modified to remove or duplicate one or more of the endogenous alternates to increase or decrease a number transgene of interest substitutions that replace the endogenous alternates in the alternatively spliced cassette section.

9. A plurality of proteins that has been produced by performing the method according to any one of claims 1 to 8.

10. A mutually-exclusive poly transgene expression system comprising: an upstream nucleotide sequence; a downstream nucleotide sequence; and an alternatively spliced cassette section located between the upstream nucleotide sequence and the downstream nucleotide sequence, wherein the alternatively spliced cassette section comprises: a plurality of transgenes of interest, wherein each transgene of interest of the plurality of transgenes of interest is substituting one of a plurality of endogenous alternates; and intron sequences located between the transgenes of interest of the plurality of transgenes of interest.

11. The mutually-exclusive poly transgene expression system according to claim 10, wherein the alternatively spliced cassette section is derived from a Drosophila Down syndrome cell adhesion molecule 1 gene.

12. The mutually-exclusive poly transgene expression system according to claim 10, wherein the alternatively spliced cassette section is derived from Exon 4 of the Drosophila Down syndrome cell adhesion molecule 1 gene.

13. The mutually-exclusive poly transgene expression system according to any one of claims 10 to 12, wherein the upstream genetic sequence has been modified to include at least one mutationin each start codon present in a native genetic sequence from which the upstream genetic sequence has been derived.

14. A cell that has been transfected with the mutually-exclusive poly transgene expression system according to any one of claims 10 to 13.

15. A transgenic arthropod that is constituted at least in part from the cells according to claim 14.

16. A method of generating a mutually-exclusive poly transgene expression system, comprising replacing endogenous alternates of an alternatively spliced cassette section with transgenes of interest, wherein each of the endogenous alternates is replaced by one of the transgenes of interest.

17. The method according to claim 16, wherein the endogenous alternates include all of the endogenous alternates of the alternatively spliced cassette section.

18. The method according to claim 16 or claim 17, further comprising: identifying start codons in a genetic sequence upstream from the alternatively spliced cassette; and inserting point mutations in a subset of the start codons.

19. The method according to any one of claims 16 to 18, further comprising removing or duplicating one or more of the endogenous alternates in order to decrease or increase a number of transgenes of interest that can be accommodated by the alternatively spliced cassette section.

20. The method according to any one of claims 16 to 19, wherein the alternatively spliced cassette section is Exon 4 of a Drosophila Down syndrome cell adhesion moleculel gene.

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