WO2018228961A1

WO2018228961A1 - Genetic tools and procedure for the phenotypic identification of the genotype of transgenic diploid organisms

Info

Publication number: WO2018228961A1
Application number: PCT/EP2018/065262
Authority: WO
Inventors: Frederic STROBL; Ernst H.K. Stelzer
Original assignee: Johann Wolfgang Goethe-Universität Frankfurt am Main
Priority date: 2017-06-12
Filing date: 2018-06-11
Publication date: 2018-12-20

Abstract

Diploid transgenic animals are either hemizygous or homozygous. Genetic assays are, therefore, necessary to identify the genotype. Herein disclosed is a novel vector system, referred to as the"AGameOfClones" vector concept, which uses two transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to heterozygous progeny that is identifiable by both markers. In the following generation, the omission of one marker indicates homozygous individuals. The inventors prove this concept in Tribolium castaneumby systematically creating multiple homozygous transgenic lines suitable for long-term fluorescence live imaging. Since this approach relies on the universal Cre-Lox system, it should work in most diploid model organisms, e.g. rodents, zebrafish, insects and plants. It saves resources, simplifies transgenic animal handling and contributes to the ethically motivated endeavor to minimize the number of wasted animals.

Description

GENETIC TOOLS AND PROCEDURE FOR THE PHENOTYPIC IDENTIFICATION OF THE GENOTYPE OF TRANSGENIC DIPLOID ORGANISMS

FIELD OF THE INVENTION

Diploid transgenic animals are either hemizygous or homozygous. Genetic assays are, therefore, necessary to identify the genotype. Herein disclosed is a novel vector system, referred to as the "AGameOfClones vector system", which uses two transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to heterozygous progeny that is identifiable by both markers. In the following generation, the omission of one marker indicates homozygous individuals. The inventors prove this concept in Tribo- lium castaneum by systematically creating multiple homozygous transgenic lines suitable for long-term fluorescence live imaging. Since this approach relies on the universal Cre-Lox system, it should work in most diploid model organisms, e.g. rodents, zebrafish, insects and plants. It saves resources, simplifies transgenic animal handling and contributes to the ethically motivated endeavor to minimize the number of wasted animals.

DESCRIPTION

Life sciences, especially cell and developmental biology, rely on model organisms. The most frequently used vertebrates are mouse (Mus musculus) and zebrafish {Danio rerio). Amongst insects, fruit fly (Drosophila melanogaster) and red flour beetle {Tribolium castaneum) are the two main species. One of the most important standard techniques is transgenesis, i.e. the insertion of recombinant DNA into the genome of the model organism (Gama Sosa et al., 2010). Transgenesis is mainly used in two experimental strategies: Firstly, knock-in assays, where the transgene usually contains a functional expression cassette. Secondly, knock-out assays, where endogenous genes or genetic elements are rendered inoperative. Since model organisms are typically diploid, the genotype has to be considered, which leads to a certain experimental complexity. Usual crossing setups can result in (i) non-transgenic wild-type animals, (ii) hemizygous transgenic animals, i.e. only the maternal or the paternal chromosome carries the transgene, and (iii) homozygous transgenic animals, i.e. both the maternal and paternal chromosomes carry the transgene. In some cases, the phenotype will reveal the genotype, but usually, either two of the three or even all three outcomes cannot be identified. Transformation markers can be used to separate wild-type from transgenic animals, but do not allow to distinguish between hemizygotes and homozygotes. Thus, additional experi- ments are necessary to determine the genotype, for example genetic assays, which are invasive and require manpower, consumables and time.

In the AGameOfClones (AGOC) vector system, all genotypes are identifiable by purposely produced distinct phenotypes, which permits the systematic creation of homozygous transgenic animals. The concept relies on two phenotypically clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination results in hemizygous animals that retain only one of both markers. Thus, they can be phenotypically distinguished from each other and from the wild-type. In the next generation, individuals that express both markers are identified as heterozygous for the transgene. Finally, a cross of two heterozygotes results in homozygous progeny that is selected by the omission of one marker.

It was therefore an objective of the present invention to provide novel means to facilitate molecular genetic manipulations of organisms, in particular the introduction of sequence alterations such as transgenic sequences into diploid organisms.

The above problem is solved in a first step by a vector system comprising nucleic acid sequences encoding several different transgenes, characterized in that nucleic acid sequences encoding a transgene, each comprise autonomous nucleic acid sequences controlling the expression of the respective transgene,

wherein each nucleic acid sequence encoding a transgene together with its autonomous nucleic acid sequence controlling the expression of the respective transgene comprises donor/acceptor-nucleic acid sequences at the proximal 5'-end and at the proximal 3'-end which can be recognized by a recombination enzyme,

wherein the 5' donor nucleic acid sequence (a) which is located proximally to the s'- end of the nucleic acid sequence of a first transgene and the 5' acceptor-nucleic acid sequence

(c) which is located proximally to the s'-end of the nucleic acid sequence of a further transgene can be recognized and recombined by a recombination enzyme, and

wherein the 3' donor nucleic acid sequence (b) which is located proximally to the s'- end of the nucleic acid sequence of a first transgene and the 3' acceptor-nucleic acid sequence

(d) which is located proximally to the 3'-end of the nucleic acid sequence of a further transgene can be recognized and recombined by a recombination enzyme.

The vector system of the invention is herein also often referred to as "AGameOfClones vector system". Generally, a vector system of the invention comprises at least one nucleic acid construct having any selection of: expressible sequences, enzyme recognition sites, promoter elements, enhancer elements, transcriptional start sites, and/or gene sequences. The vector system may also include any sequence element known in the art and used for molecular genetic experimentation. Hence, a "vector system" according to the invention may comprise one more single vector(s), in which in the recited sequence elements of the invention are distributed on the one or more single vector(s).

As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting one or more other nucleic acid sequence(s) to which it has been linked or which was introduced into said vector. One type of vector is a "plasmid", which refers to a circular double stranded DNA into which additional DNA segments may be cloned. Another type of vector is a viral vector, wherein additional DNA segments may be cloned into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

The vector may also contain additional sequences, such as a polylinker for subcloning of additional nucleic acid sequences, or a polyadenylation signal to effect proper polyadenylation of the transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the methods of the disclosure, and any such sequence may be employed, including but not limited to the SV40 and bovine growth hormone poly-A sites. Also contemplated as an element of the vector is a termination sequence, which can serve to enhance message levels and to minimize readthrough from the construct into other sequences. Additionally, expression vectors typically have selectable markers, often in the form of antibiotic resistance genes, that permit selection of cells that carry these vectors.

In one embodiment of the invention the vector system disclosed herein is characterized in that it comprises the following nucleic acid sequences in 5' to 3' direction:

(i) a recombinase-donor nucleic acid sequence (a) which is located proximally to the 5'-end of a first transgene, (ii) a nucleic acid sequence encoding a first transgene and its autonomous expression control sequence (preferably upstream of the first transgene),

(iii) a recombinase-donor nucleic acid sequence (b) which is located proximally to the 3'-end of a first transgene,

iv) a recombinase-acceptor nucleic acid sequence (c) which is located proximally to the 5'-end of a further transgene,

(v) a nucleic acid sequence encoding a further transgene and its autonomous expression control sequence (preferably upstream of the further transgene), and

(vi) a recombinase-acceptor nucleic acid sequence (d) which is located proximally to the 3'-end of a further transgene.

In some embodiments the vector system comprises two nucleic acid sequences encoding two different transgenes, wherein preferably the two different transgenes have non-identical sequences. Even more preferably the transgenes encode different (or non-identical) polypeptide sequences.

In some preferred embodiments of the invention the polypeptide sequences are polypeptide sequences of biomarkers. The term "bio marker" shall refer in context of the invention to any polypeptide that when introduced into a diploid organism is detectable by any means. Such biomarkers usually comprise polypeptides that when expressed confer a particular phenotyp- ic change in the organism. Many such genetic markers are known for any given genetic model organisms. A preferred biomarker of the invention is a fluorescent or luminescent or other optically detectable marker. Other examples of genetics markers are fur color markers for mouse (Zheng et al. 1999, Nucleic Acids Research), eye pigmentation marker in Drosophila melanogaster (Rubin and Spradling 1982, Science) and Tribolium castaneum (Lorenzen et al. 2002, Genetics), cuticule pigmentation markers for insects in general (Osanai-Futahashi et al. 2012, Nature Communications) and beta-glucoronidase-based markers for Plants (George Acquahh, Principles of Plant Genetics and Breeding, Wiley- Blackwell 2007).

The term "recombinase" as used herein refers to a group of enzymes that can facilitate recombination, preferably, site specific recombination, between defined sites, called "recombination sites", where the two recombination sites are physically separated within a single nucleic acid molecule or on separate nucleic acid molecules. The sequences of the two defined recombination sites are not necessarily identical. Within the group of recombinases there are several subfamilies including "integrases" (for example, like Cre, Cre-like, FLP and λ inte- grase), "resolvases/invertases" (for example, c C3i integrase, R4 integrase, and TP-901 inte- grase). The term "recombinase" also includes, but is not limited to, prokaryotic or eukaryotic transposases, viral or Drosophila copza-like or non-viral reterotransposons that include mammalian reterotransposons. Exemplary prokaryotic transposases include transposases encoded in the transposable elements of Tni, Tn2, Tn3, Tn4, Ή15, Tn6, Tn9, Tnio, Tn30, Tnioi, Tn50i, Tn903, Tniooo, Tni68i, Tn290i, etc. Eukaryotic transposases include transposases encoded in the transposable elements of Drosophila mariner, sleeping beauty trans- posase, Drosophila P element, maize Ac and Ds elements, etc. Retrotransposases include those encoded in the elements of Li, T0I2 Tci, Tc3, Mariner (Himar 1), Mariner (mos 1), Minos, etc. Transposases may also be selected from Mp, Spm, En, dotted, Mu, and I transposing elements. Preferred is however that the donor-/ acceptor nucleic acid sequences can be recognized and recombined by the Cre-recombinase.

As used herein, the term "Cre recombinase" refers to the site-specific DNA recombinase derived from the Pi bacteriophage. Cre recombinase is a 38-kDa product of the cre (cyclization recombination) gene of bacteriophage Pi and is a site-specific DNA recombinase of the nt family (Sternberg, N. et al. (1986) J. Mol. Biol. 187:197-212). Cre recombinase recognizes a 34-bp site on the Pi genome called LoxP (locus of X-over Pi, LoxP site) and efficiently catalyzes reciprocal conservative DNA recombination between pairs of LoxP sites. The LoxP site comprises two 13-bp inverted repeats flanking an 8-bp nonpalindromic core region. Cre re- combinase-mediated recombination between two directly repeated LoxP sites results in excision of DNA between. The Cre recombinase is known in the art. See, for example, Guo et al. (1997) Nature 389:40-46; Abremski et al. (1984) J. Biol. Chem. 259:1509-1514; Chen et al. (1996) Somat. Cell Mol. Genet. 22:477-488; and Shaikh et al. (1977) J. Biol. Chem. 272:5695- 5702. The Cre recombinase will recognize a number of variant or mutant lox sites relative to the LoxP sequence. Conversely, the LoxP sequence can be recognized by mutants and variants of Cre recombinase. Examples of these variant or mutant Cre recombination sites include, but are not limited to, the LoxB, LoxL and LoxR sites which are found in the E. coli chromosome (Hoess et al. (1982) Proc. Natl. Acad. Sci. USA 79:3398). Other variant lox sites include: L0XP511 site (5 '-ATAACTTCGTATAGTATACATTATACGAAGTTAT-3 ' (Hoess et al. (1986) Nucleic Acid Res. 14:2287-2300)) and LoxC2 site (5'- ACAACTTCGTATAATGTATGCTATACGAAGTTAT-3' (U.S. Pat. No. 4,959,317)). Also possible is the use of LoxN sites as for example disclosed in Livet J et al: "Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system" (Nature. 2007 Nov i;450(7i66):56-62). Therefore, the term "Cre recombinase" includes Cre recombinase and all mutants and variants thereof with the recombination catalyzing function described above, and the term "LoxP site" includes the 34 bp site described above and any mutants and variants recognizable by the Cre recombinase, including its variants. An alternative recombinase is a FLP recombinase. As used herein, the term "FLP recombinase" refers to a site-specific DNA recombinase derived from yeast, the 2pi plasmid of Saccharomyces cerevisiae, that recognizes a 34 base pair DNA sequence, termed the "FRT site" (FLP recombinase target). "FLP" is a 423 amino acid protein capable of binding to FRT recombination target sites and mediating conservative site-specific recombination between FRT sites. The basic configuration of the FRT site comprises a 48 nucleotide DNA sequence consisting of an 8-base-pair core and three 13-base-pair symmetry elements where two symmetry elements occur in direct orientation on the 5' end of the core sequence and the third element occurs in inverted orientation on the 3' end of the core sequence. The FRT site has also been identified as minimally comprising two 13 base-pair repeats, separated by an 8 base-pair spacer, as follows: 5'- GAAGTTCCTATTC [TCTAGAAA] GTATAGGAACTTC-3 ' . The nucleotides in the above "spacer" region can be replaced with any other combination of nucleotides, so long as the two 13 base-pair repeats are separated by 8 nucleotides. The actual nucleotide sequence of the spacer is not critical, although those of skill in the art recognize that, for some applications, it is desirable for the spacer to be asymmetric, while for other applications, a symmetrical spacer can be employed. It is also recognized by one skilled in the art that modified forms or mutants of FLP recombinase can recognize the FRT site and its variants. Therefore, the term "FLP recombinase" and "FRT site" includes all variants and mutants carrying out their functions as described above.

In some embodiments it is preferred that the donor-/ acceptor nucleic acid sequences which can be recognized and recombined by the Cre-recombinase, are lox sequences.

In some embodiments, the vector system according to the invention is characterized in that the 5' donor nucleic acid sequence (a) which is located proximally to the s'-end of the nucleic acid sequence of a first transgene and the 5' acceptor-nucleic acid (c) which is located proximally to the s'-end of the nucleic acid sequence of a further transgene, are lox sequences which are different from the 3' donor nucleic acid sequence (b) which is located proximally to the 3'-end of the nucleic acid sequence of a first transgene and the 3' acceptor-nucleic acid (d) which is located proximally to the 3'-end of the nucleic acid sequence of a further transgene.

In some embodiments, the vector system according to the invention is characterized in that the donor-/ acceptor nucleic acid sequences (a) and (c) are either LoxP- or LoxN nucleic acid sequences.

In some embodiments, the vector system according to the invention is characterized in that the donor-/ acceptor nucleic acid sequences (b) and (d) are either LoxP- or LoxN nucleic acid sequences.

In some embodiments, the vector system according to the invention is characterized in that the vector system can be introduced/integrated in the germ line. In some embodiments, the vector system according to the invention is characterized in that it comprises the sequence(s) according SEQ ID NO: l or 2.

In some embodiments, the vector system according to the invention is characterized in that it is a shuttle vector system. In other embodiments the vector system of the invention is a vector system for use in a method of establishing a transgenic organism.

In some embodiments of the invention the vector system described herein is characterized in that it comprises a nucleic acid sequence encoding recombinase as described before, preferably a Cre-recombinase.

In some embodiments of the invention the vector system described herein is characterized in that it comprises a nucleic acid sequence encoding a Cre-recombinase, wherein it comprises a further nucleic acid sequence controlling the inducible expression of the Cre-recombinase.

The object of the invention is also solved in an additional aspect by a cell, characterized in that the cell comprises a vector system as disclosed herein. Preferably the cell is a biological cell, such as a eukaryotic cell, most preferably a cell derived from a plant or animal, such as an insect, a fish, a bird or a mammal. A mammal is preferably selected from the group comprising mouse, rat, guinea pig, rabbit, pig, cat, dog, goat, lama, camel, horse and monkey.

In another aspect the invention also provides a multicellular transgenic organism, characterized in that it comprises a vector system as described herein before.

A further aspect then pertains to a method for the generation of a transgenic organism, the method comprising the following steps:

(i) Introduction of a vector system as described herein into an embryo or a germ cell (of said organism),

(ii) Selection of hemizygous organisms expressing at least two of the transgenes which are encoded by the vector system,

(iii) Optionally, crossing of transgenic, non-human, multicellular hemizygous organisms derived from step (ii) with non-human, multicellular organisms expressing a recombinase transgene,

(iv) Selection of descendants/offsprings which have been obtained by crossing of hemizygous organisms according to step (ii) with non-human, multicellular organisms expressing a recombinase transgene, and a subsequent out-crossing against the wildtype, and which express only one of the at least two transgenes which are encoded by the vector system of the invention,

(v) Crossing of transgenic, non-human, multicellular hemizygous organisms which express only one of the at least two transgenes, with transgenic non-human, multicellular hemizygous organisms which express another of the at least two transgenes,

(vi) Identification of transgenic, non-human, multicellular heterozygous descendants/offsprings, which express two of the transgenes,

(vii) Crossing of transgenic, non-human, multicellular heterozygous organisms which express two of the transgenes, with transgenic, non-human, multicellular heterozygous organisms which express two of the transgenes,

(viii) Identification of transgenic, non-human, multicellular homozygous descendants/offsprings, which express (preferably only) one of the transgenes.

In some embodiments the invention provides the above method, wherein the vector system according to the invention has been introduced into an embryo or a germ cell, which is able to express the Cre-recombinase in step (i), and wherein step (iii) of the method is omitted.

In some embodiments the invention provides the above method, wherein the vector system has been introduced into an embryo or a germ cell in step (i), and wherein the procedure according to step (i), optionally, comprises the induction of the expression of a nucleic acid sequence encoding the Cre-recombinase.

In some embodiments the transgenic organism is a multicellular organism, preferably selected from a group comprising the following classes: plants or animals, such as insects, birds, fish and mammals. A mammal may be selected from a group consisting of the following members: mouse, rat, guinea pig, rabbit, pig, cat, dog, goat, lama, camel, horse and monkey.

Another aspect of the invention then pertains to a transgenic organism obtained or obtainable by a method for the generation of a transgenic organism as described herein before.

Yet a further aspect then pertains to a use of a vector system of the invention, characterized in that it is used for the identification of hemizygous, heterozygous and/or homozygous, non- human, multicellular organisms, who are derived in a method for the generation of a transgenic organism as described herein before.

The present invention will now be further described in the following examples with reference to the accompanying figures and sequences, nevertheless, without being limited thereto. For the purposes of the present invention, all references as cited herein are incorporated by reference in their entireties. In the Figures:

Figure l shows the AGameOfClones vector concept within the piggyBac-based pAGOC transformation vector for Tribolium castaneum. (A) Two fluorescence-based transformation markers, mO and mC, which are spectrally distinct, are embedded into a piggyBac-based transformation vector that is characterized by 3' and 5' terminal repeats (TR) necessary for genomic insertion. The markers are based on the artificial 3XP3 promoter, which drives expression in the neuronal system, the open reading frame for the respective fluorescence protein, i.e. mOrange or mCherry, and the SV40 poly (A) site. Each transformation marker is flanked upstream by a LoxP and downstream by a LoxN site, which results in interweaved Lox site pairs. In Tribolium castaneum adults, the fluorescence phenotype is detected in the eyes using appropriate filter sets. (B) Cre- mediated recombination leads to the excision of one of either markers from the genome. When one marker is removed, the other marker remains within the genome, since the two remaining lox sites, i.e. a LoxP and a LoxN site, are incompatible. Tribolium castaneum adults that underwent recombination give rise to progeny where only one marker is expressed in the eyes.

Figure 2 shows the AGameOfClones F3 to F7 crossing procedure that allows the systematic creation of homozygous transgenic animals. The rounded rectangle illustrates the genotype for two independent autosomes, white bars represent the AGOC transgene location and black bars the helper transgene location. A F2 mO-mC founder female x wild-type male outcross results in F3 mO-mC pre-recombination hemizygotes that carry mO and mC consecutively on one chromosome. A F3 mO-mC pre-recombination hemizygous female x mCe homozygous helper male cross gives rise to F4 mCexmO-mC double hemizygotes, in which one marker is removed through Cre-mediated recombination. A F4 mCexmO-mC double hemizygous female x wild-type male outcross generates F5 mO- and mC-only post-recombination hemizygotes. Next, a F5 mO- only post-recombination hemizygous female x F5 mC-only post- recombination hemizygous male brother-sister cross results in a certain fraction of F6 mO/mC heterozygous progeny. Finally, a F6 mO/mC heterozygous female x a F6 mO/mC heterozygous male brother-sister cross generates F7 mO- and mC-only homozygous progeny. The percentage boxes show the theoretical ratio of the progeny that carry the respective genotype. Figure 3 shows the AGameOfClones F3 to F7 crossing procedure shown for the AGOC #5 and #6 sublines. From the F3 to the F7 generation, the genotype was phe- notypically determined by monitoring mCe, mO and mC. For both sublines, F7 mO- and mC-only homozygotes were obtained by following the systematic procedure outlined in Figure 2. For each individual, a wild-type control of the same gender is shown. The percentage boxes show the experimental (and theoretical) ratios of the progeny that show the respective phenotype.

Figure 4 shows the crossing results for the six proof-of-principle AGOC lines from the

F3 to F7 generation. (1) in the AGOC #4 subline, incomplete recombination in the F4 mCexmO-mC double hemizygotes occurred, as the inventors obtained several F5 individuals that still carry both transformation markers (7% in total). The inventors continued the AGOC procedure with the F5 mO- and mC- only post-recombination hemizygous progeny.

Figure 5 shows the F4 mCexmO-mC double hemizygote generation. The F4 hybrids are hemizygous for both the AGOC transgene, which carries both mO and mC embedded into interweaving Lox site pairs, and for the helper transgene, which carries mCe and the Cre recombinase expression cassette. Within this cassette, expression of a nuclear-localized Cre recombinase is driven by the endogenous HSP68 promoter. During the development of the F4 generation from the zygote to the fertile adult, the HSP68 promoter exhibits a slight leaky expression. Over time, this leads to recombination in germ cells (i.e. the prerequisite for the AGOC vector concept) but also in certain somatic progenitor cells that later on give rise to distinct, typically spatially clustered cell populations. This effect is evident in the compound eyes of adult animals in all AGOC lines, where certain clusters of ommatidia show either expression of mO or mC.

Figure 6 shows the AGameOfClones F3 to F7 crossing procedure with inversed genders.

To prove that the proposed concept is not gender-specific, the F3 to F7 crossing procedure was carried out as described in the main text (Figure 2), but with inversed genders. F3 mO-mC pre-recombination hemizygous males of the AGOC #5 and #6 sublines were crossed against mCe helper homozygous females of the ICE{HSP68'NLS-Cre} #1 subline, resulting in F4 mCexmO-mC double hemizygotes, in which Cre-mediated recombination occurs. F4 mCexmO-mC double hemizygote males were outcrossed against wild-type females, this results in F5 mO- and mC-only post-recombination hemizygotes that carry either only mO or only mC on the paternal chromosome. F5 mO- only post-recombination hemizygous males were brother-sister crossed against F5 mC-only post-recombination hemizygous females, resulting in F6 mO/mC heterozygotes that carry once again both markers. F6 mO/mC heterozygous males were brother-sister crossed against genotypic identical F6 mO/mC heterozygous females, resulting in F7 mO- and mC-only homozygotes that carry either only the mO or only the mC marker on both, the maternal and paternal chromosomes. Similar to the standard, the procedure could be performed also successfully with inversed genders. For each individual, a wild- type control of the same gender is shown. The percentage boxes show the experimental (and theoretical) ratios of the progeny that show the respective phenotype.

Figure 7 shows the AGameOfClones F3 to F7 crossing procedure with a different mCe helper subline. To prove that the proposed concept does not rely solely on one Cre recombinase expressing helper subline, the F3 to F7 crossing procedure was carried out as described in the main text (Figure 2), but with the ICE{HSP68'NLS-Cre} #2 helper subline, which carries the same transgene as #1 subline, but at a different genomic location. The subline is homozygous for the transgene. F3 mO-mC pre-recombination hemizygous females of the AGOC #5 and #6 sublines were crossed against mCe helper homozygous males of the ICE{HSP68'NLS-Cre} #2 subline, resulting in F4 mCexmO-mC double hemizygotes, in which Cre-mediated recombination occurs. F4 mCexmO-mC double hemizygote females were outcrossed against wild-type males, this results in F5 mO- and mC-only post-recombination hemizygotes that carry either only mO or only mC on the paternal chromosome. F5 mO-only post- recombination hemizygous females were brother-sister crossed against F5 mC- only post-recombination hemizygous males, resulting in F6 mO/mC heterozygotes that carry once again both markers. F6 mO/mC heterozygous females were brother-sister crossed against genotypic identical F6 mO/mC heterozygous males, resulting in F7 mO- and mC-only homozygotes that carry either only the mO or only the mC marker on both, the maternal and paternal chromosomes. Similar to the standard, the procedure could be performed also successfully with inversed genders. For each individual, a wild-type control of the same gender is shown. The percentage boxes show the experimental (and theoretical) ratios of the progeny that show the respective phenotype.

Figure 8 shows fluorescence live imaging of selected homozygous functional AGOC sublines. (A) An AGOC{Zeni'#0(LA)-mEmerald} #2 embryo during gastrulation. This subline permits the characterization of actin and actomyosin dynamics that are involved in serosa window closure (first and second row). It can also be used to describe the cytoskeleton rearrangement of the dorsal blastoderm cells and to analyze their appearance change during differentiation to serosa cells (third row and detail images). (B) An AGO CXARP5 '# O (LA) -mEmerald} #1 embryo during germband retraction. In this subline, the brain and ventral nerve cord expresses Lifeact-mEmerald on a high level, permitting the observation of neurulation. Detail images show the forming ganglia of the first and second thoracic segment. (C) Optical sections of an AGOC{ARPs'#0(LA)- mEmerald} #i embryo after dorsal closure. Detail images show the supra- and suboesophageal ganglia. (D) Comparison of embryos from the AGOC{ARP5'#0(LA)-mEmerald} #1 and #2 sublines after dorsal closure. In contrast to the expression level in the #i subline, the Lifeact-mEmerald expression level of the AGOC{ARP5'#0(LA)-mEmerald} #2 subline in the nervous system is very low. ZP, z maximum projection with image processing; ZA, z maximum projection with intensity adjustment; PA, single plane with intensity adjustment.

Figure 9 shows the pAGOC vector. (A) Vector map of pAGOC, which is based on the pAVOIAF[#i-#2-#3-#4] vector. In this vector, #1 and #2 remain empty, while mO and mC together with their flanking upstream LoxP and downstream LoxN sites were inserted into #3 and #4, respectively. The non- annotated dark blue boxes represent the same restriction enzyme sites as shown for the pAVOIAF[#i-#2-#3-#4] vector. The light gray band on the inside indicates the transgene. (B) Scheme of mO and mC that are embedded into intermeshing but incompatible LoxN and LoxP site pairs. Restriction enzyme sites are not shown. Sizes of genetic elements are not to scale. ORF, open reading frame; TR, piggyBac terminal repeat.

Figure 10 shows the pAGOC{#P'#0(LA)-mEmerald} vector. (A) Vector map of pA- GOC{#P'#0(LA)-mEmerald}, which is based on the pAGOC vector (Supplementary Figure 8). In this vector, #1 remains empty, while the #P'#0(LA)- mEmerald two-slot cloning site was inserted into #2. The non-annotated dark blue boxes represent the same restriction enzyme sites as shown for the pAVOIAF[#i-#2-#3-#4] vector in Supplementary Figure 7 as well as several new restriction enzyme sites shown in (B). The light gray band on the inside indicates the transgene. (B) Scheme of the #P'#0(LA)-mEmerald two-slot cloning site. To insert a promoter, the #P slot can be accessed by the Ascl/Fsel restriction enzyme site pair, but alternatively by the double BtgZI restriction enzyme site pair, which flanks a FREDDY spacer. BtgZI is a type I restriction enzyme with a non-palindromic recognition sequence. It cuts the sequence several bp (10/14) downstream, resulting in a 4 bp sticky end. In this vector, the upstream BtgZI restriction enzyme site (in reverse orientation) allows the opening of the Ascl restriction enzyme site, while the downstream BtgZI restriction enzyme site (in forward orientation) allows the opening of the Lifeact open reading frame start codon and the first bp of the subsequent codon, which allows scarless insertion of respectively digested promoter sequences (indicated by arrows). The Lifeact (LA) open reading frame, which is in #0 per default, can be substituted with another open reading frame to change the intracellular localization by the Fsel/NotI restriction enzyme site pair, while the mEmerald open reading frame can be substituted with another fluorescence protein open reading frame by the Notl/Sbfl restriction enzyme site pair. ORF, open reading frame.

Figure 11 shows nine different genetic options to create an AGOC vector. The first variant is identical to the variant presented in Figure 1. Variant 1 to 5 are based on recombination-excision, while variants 6 to 9 are based on recombination- inversion. Promoter 1 and Promoter 2 resemble two regulatory sequences with a different spatiotemporal expression pattern. G and R resemble two different polypeptide sequences, for example a green and a red fluorescent protein. Pre- R, pre-recombination; Post-R, post-recombination.

EXAMPLES

Materials and Methods

Molecular biology: the pUCrAGOCI and pAGOC vectors

The 5,678 bp pUC57[AGOC] vector was ordered as a de novo gene synthesis construct (Ge- newiz). A 3,277 bp insert was inserted into the unique Ndel and Pstl restriction enzyme sites of pUC57-Kan, it consists of (i) the 4 bp TTAA 3' piggyBac excision/insertion target sequence, (ii) the 235 bp piggyBac 3' terminal repeat, (iii) the 2,724 bp four-slots (#1 to #4) cloning site, in which the mOrange-based and mCherry-based eye-specific transformation markers (mO and mC, respectively) were already inserted (both in reverse orientation and thus tail-to- head) into #3 and #4, (iv) the 310 bp 5' piggyBac terminal repeat and (v) the 4 bp TTAA 5' piggyBac excision/insertion target sequence. The insert was amplified with a respective primer pair, which introduced upstream an Aatll and downstream a Pcil restriction enzyme site. The PCR product and PUC57LAGOC] were digested accordingly, and the insert was reintegrated into the vector, reducing the vector size considerably by removing 629 functionless bp. The resulting 5,049 bp vector was termed pAGOC (Figure 9) and used (i) for germline transformation to create proof-of-principle lines, and (ii) as an intermediate vector for further cloning operations.

Molecular biology: the pGSr#P'#Q(LA)-mEmeraldl and pAGOC{#P'#Q(LA)-mEmerald> vectors

The 4,111 bp the pGS[#P'#0(LA)-mEmerald] vector was ordered as a de novo gene synthesis construct (Thermo Fisher Scientific). A 1,821 bp insert was inserted into the unique Sfil restriction enzyme site of pMK-RQ (Thermo Fisher Scientific), it consists of (i) a 6 bp Hindlll restriction enzyme site, (ii) the 107 bp #P'#0 two-slot subcloning site, (iii) a 9 bp linker that includes a Notl restriction enzyme site, (iv) the 714 bp codon-optimized mEmerald open reading frame, (v) a 8 bp Sbfl restriction enzyme site, (vi) a 983 bp elongated variant of the SV40 poly(A) site and (vii) a 6 bp Xbal restriction enzyme site. The insert was cut from the backbone with Hindlll / Xbal and pasted into #3 of the pAGOC vector. The resulting 6,852 bp vector was termed pAGOC{#P'#0(LA)-mEmerald} (Figure 10) and used as an intermediate vector for further cloning operations.

Molecular biology: the promoter library vectors

All library vectors are based on pGEM-T Easy (A1360, Promega). The two promoter library vectors were created by amplifying the sequences from genomic DNA with the respective extraction PCR primer pairs. Amplification was followed by A-tailing using the Recombinant Taq DNA polymerase (10342020, Thermo Fisher Scientific) and subsequent ligation into pGEM-T Easy. The resulting vectors were termed pTC-Zeni'-GEM-T Easy and pTC-ARPs'- GEM-T Easy and had sizes 4,599 bp and 5,516 bp, respectively.

Molecular biology: the pAGOGiZeni'#Q(LA)-mEmerald} and pAGOGiARPf;'#0(LA)- mEmerald} vectors

Two transformation vectors were created that allowed the expression of mEmerald-labeled Lifeact (LA) in different spatiotemporal patterns. Therefore, the promoter sequences were amplified from the library vectors with the respective primer pairs, which introduced upstream an Ascl and downstream a Bsal restriction enzyme site. The PCR product was digested accordingly, and the pAGOC{#P'#0(LA)-mEmerald} vector was digested with BtgZI, which led to compatible overhangs and allowed scarless insertion of the promoter sequences into the #P slot. The resulting vectors were termed pAGOC{Zeni'#0(LA)-mEmerald} and pAGOC{ARP5'#0(LA)-mEmerald}, had sizes of 8,367 bp and 9,276 bp, respectively, and were used for germline transformation.

Germline transformation of Tribolium castaneum

Approximately 500-600 Fo Tribolium castaneum adults of the PWAS strain were incubated on 405 fine wheat flour (113061036, Demeter, Darmstadt, Germany) supplemented with 5% (wt/wt) inactive dry yeast (62-106, Flystuff, San Diego, CA, USA) at 25°C and 70% relative humidity in light for 2 h. After the incubation period, the adults were removed and the embryos (around 700-900) were extracted from the flour and incubated another hour as stated above. Afterwards, they were transferred to a 100 μηι cell strainer (352360, BD Biosciences) and washed in a 6-well plate as follows: (i) in autoclaved tap water for 1 min, (ii) in 10% (vol/vol) sodium hypochlorite (425044-250ML, Sigma Adlrich) in autoclaved tap water for 10 s, (iii) in autoclaved tap water three times for 1 min and (iv) stored in autoclaved tap water. Next, the embryos were lined up on object slides within the next hour (10 object slides with about 50 embryos reach, totaling at approximately 500 embryos per round of germline transformation) with their posterior pole pointing towards the elongated edge of the slide. The embryos were injected with a mixture of 500 ng/ μΐ transformation vector and 400 ng/ μΐ transposase-expressing helper vector (pATub'piggyBac) in injection buffer (5 mM KC1, 1 mM KH2PO4 in ddH20, pH 8.8). For injection, a microinjector (FemtoJet, Eppendorf) and 0.7 μηι outer diameter capillaries (Femtotips II, Eppendorf) with an injection pressure of 400- 800 hPa were used. After injection, the object slides with embryos were placed in Petri dishes on a 5 mm 1% (wt/vol) agarose in tap water 'platform' and incubated at 32°C. After 3 days, hatched larvae, i.e. Fi potential mosaics, were collected and raised individually in single wells of 24-well plates as described above.

Light sheet-based fluorescence microscopy Three Tribolium castaneum sublines were characterized with long-term fluorescence live imaging, the AGOC{Zeni'#0(LA)-mEmerald} #2 subline, which exhibited the strongest fluorescence signal out of the three sublines, and both AGOC{ARP5'#0(LA)-mEmerald} sublines, since they showed a slightly varying expression pattern. Fluorescence live imaging was performed with digitally scanned laser light sheet-based fluorescence microscopy. In brief, embryo collection was performed with the F7+ continuative mC-only homozygous cultures for one hour at 25°C, and embryos were incubated for 15 hours at 25°C. Sample preparation took approximately one hour at room temperature (23±i°C), so that embryos were at the beginning of gastrulation, when the uniform blastoderm turns into the rearranged blastoderm. Embryos were recorded along four directions (in the orientations 0°, 90⁰, 180⁰ and 270⁰) with an interval of 30 minutes for up to four days at room temperature, covering the embryogenetic events gastrulation and germband elongation completely and germband retraction partially. All embryos survived the imaging procedure, developed to healthy and fertile adults, and when outcrossed

Example 1: Proof-of-principle in the emerging insect model organism Tribolium castaneum

The proof-of-principle of the AGOC vector concept relied on the red flour beetle Tribolium castaneum (Brown et al., 2009), an emerging insect model organism, in conjunction with the piggyBac transposon system (Lorenzen et al., 2003), which allows semi-random genomic insertion. The inventors created a piggyBac-based transformation vector termed pAGOC, which contains a mOrange-based (Shaner et al., 2008) and a mCherry-based (Shaner et al., 2004) eye-specific (Wimmer et al., 1999) transformation marker, termed mO and mC, respectively. Both fluorescent proteins are spectrally separable by appropriate excitation and emission. Each transformation marker is flanked upstream by a LoxP site (Hamilton and Abremski, 1984) and downstream by a LoxN (Livet et al., 2007) site, resulting in inter- weaved, but incompatible Lox site pairs (Figure 1).

The inventors injected this vector together with a piggyBac transposase-expressing helper vector (pATub'piggyBac) into pre-blastoderm embryos to achieve germline transformation. All survivors, termed Fi potential mosaics, were outcrossed against the wild-type. In six of those crosses, one or more F2 mO-mC hemizygous founder females were found among the progeny, which were outcrossed against wild-type males. Transgenic progeny was collected to establish six proof-of-principle cultures, which carry the same transgene, but in different genomic locations. These F3 mO-mC pre-recombination hemizygous sublines were termed AGOC #1 to #6. Until this step, this route did not differ from most standard transgenic animal establishment procedures. The systematic creation of homozygous transgenic animals (Figure 2) was performed with all six AGOC sublines and phenotypically documented for #5 and #6 (Figure 3). This procedure is completed within only four further generations.

(1) F3 mO-mC pre-recombination hemizygous females, which carry mO and mC consecutively on the maternal chromosome, were crossed against mCe homozygous helper males of the ICE{HSP68'NLS-Cre} #1 subline (Figure 2 and Figure 3, first row). This subline expresses a nuclear-localized Cre recombinase (Peitz et al., 2002) under control of the HSP68 promoter and carries a mCerulean-based (Markwardt et al., 2011) eye-specific transformation marker (mCe), resulting in F4 mCexmO-mC double hemizygotes, in which Cre-mediated recombination occurs (Figure 4, 'F3' row). In this hybrid generation, adult beetles show a patchy expression of both markers within their compound eyes (Figure 5).

(2) F4 mCexmO-mC double hemizygous females were outcrossed against wild-type males (Figure 2 and Figure 3, second row). Due to the Cre-mediated recombination in germline cells, this results in F5 mO- and mC-only post-recombination hemizygotes that carry either mO or mC on the maternal chromosome (Figure 4, 'F4' row).

(3) F5 mO-only post-recombination hemizygous females were brother-sister crossed against F5 mC-only post-recombination hemizygous males (Figure 2 and Figure 3, third row), resulting in F6 mO/mC heterozygotes, which carry once again both markers (Figure 4, 'F5' row). In contrast to the F3 mO-mC pre-recombination hemizygotes, which show the same phenotype but carry both markers consecutively on the maternal chromosome, the F6 mO/mC heterozygotes carry mO on the maternal and mC on the paternal chromosome. This was proven by crossing F6 mO/mC heterozygous females against wild-type males (Figure 4, 'F6-S' row).

(4) F6 mO/mC heterozygous females were brother-sister crossed against genotypic identical F6 males (Figure 2 and Figure 3, fourth row), resulting in F7 mO- and mC-only homozygotes that carry either only mO or only mC on both, the maternal and paternal chromosomes (Figure 4, 'F6' row). In contrast the F5 mO- and mC-only post-recombination hemizygotes, which show the same phenotype but carry either mO or mC on the maternal chromosome, the F7 mO- and mC-only homozygotes carry the respective marker on the maternal and paternal chromosome (Figure 2 and Figure 3, fifth row). This was proven by crossing F7 mO- and mC- only homozygous females against wild-type males (Figure 4, 'F7-O' and 'F7-C row, respectively). Although some phenotype distributions differed from the theoretical Mendelian values, all expected phenotypes, and thus all expected genotypes, were found in all generations, and both, mO- and mC-only homozygotes, could be obtained for all six AGOC sublines in the F₇. Two controls were performed with the AGOC #5 and #6 sublines to confirm proper functionality of the AGameOfClones vector concept: (i) the F3 to F7 crossing procedure was successfully conducted with inversed genders (Figure 6) and (ii) an alternative mCe helper homozygous subline, ICE{HSP68'NLS-Cre} #2, was used, which carries the same Cre-expressing cassette as the #1 subline, but at a different genomic location (Figure 7).

Example 2: Creation and live imaging of functional AGOC lines

The inventors expanded the pAGOC vector concept by creating two vectors that contain expression cassettes for mEmerald-labeled Lifeact under control of either the zerknilllt 1 promoter or the actin related protein 5 promoter. With those vectors, two transgenic Tribolium castaneum lines (with three and two sublines, respectively) were created, which are primarily designed for fluorescence live imaging of embryonic development. For all of those sublines, the systematic creation of homozygous animals was successful. The inventors performed long-term fluorescence live imaging of the embryonic development (Strobl and Stelzer, 2016) with three of the homozygous functional AGOC sublines by using a digitally scanned laser light sheet-based fluorescence microscope (Keller et al., 2008). The AGOC{Zeni'#0(LA)- mEmerald} #2 subline allows the characterization of actin dynamics within the extraembryonic membranes during gastrulation, visualizing the actomyosin cable that closes the serosa window (Figure 8A). The AGOC{ARP5'#0(LA)-mEmerald} #1 subline exhibits strong expression in the brain and ventral nerve cord and moderate fluorescence throughout the remaining embryonic tissue (Figure 8B and C). In contrast, the AGOC{ARP5'#0(LA)- mEmerald} #2 subline does not show any signal in the neuronal system but uniform fluorescence signal during germband elongation, germband retraction and dorsal closure(Figure 8C).

Claims

1. A vector system comprising nucleic acid sequences encoding several different transgenes, characterized in that nucleic acid sequences encoding a transgene, each comprise autonomous nucleic acid sequences controlling the expression of the respective transgene,

wherein each nucleic acid sequence encoding a transgene together with its autonomous nucleic acid sequence controlling the expression of the respective transgene comprises donor-/ acceptor nucleic acid sequences at the proximal s'-end and at the proximal 3'-end which can be recognized by a recombination enzyme, wherein the 5' donor nucleic acid sequence (a) which is located proximally to the s'-end of the nucleic acid sequence of a first transgene and the 5' acceptor-nucleic acid sequence (c) which is located proximally to the s'-end of the nucleic acid sequence of a further transgene can be recognized and recombined by a recombination enzyme, and

wherein the 3' donor nucleic acid sequence (b) which is located proximally to the 3'-end of the nucleic acid sequence of a first transgene and the 3' acceptor-nucleic acid sequence (d) which is located proximally to the 3'-end of the nucleic acid sequence of a further transgene can be recognized and recombined by a recombination enzyme.

2. The vector system according to claim 1, characterized in that it comprises the following nucleic acid sequences in 5' to 3' direction:

(i) a recombinase-donor nucleic acid sequence (a) which is located proximally to the 5'-end of a first transgene,

(ii) a nucleic acid sequence encoding a first transgene and its autonomous expression control sequence,

(iv) a recombinase-acceptor nucleic acid sequence (c) which is located proximally to the 5'-end of a further transgene,

(v) a nucleic acid sequence encoding a further transgene and its autonomous expression control sequence, and

3. The vector system according to claim 1 or 2, characterized in that it comprises two nucleic acid sequences encoding two different transgenes.

4. Vector system according to any one of claims 1 to 3, characterized in that the transgenes encode polypeptide sequences of different biomarkers.

5. Vector system according to any one of claims 1 to 4, characterized in that the 5' donor nucleic acid sequence (a) which is located proximally to the s'-end of the nucleic acid sequence of a first transgene and the 5' acceptor-nucleic acid (c) which is located proximally to the s'-end of the nucleic acid sequence of a further transgene, are lox sequences which are different from the 3' donor nucleic acid sequence (b) which is located proximally to the 3'-end of the nucleic acid sequence of a first transgene and the 3' acceptor-nucleic acid (d) which is located proximally to the 3'-end of the nucleic acid sequence of a further transgene.

6. Vector system according to any one of claims 1 to 5, characterized in that the donor- /acceptor nucleic acid sequences (a) and (c) are either LoxP- or LoxN nucleic acid sequences, and/or characterized in that the donor-/ acceptor nucleic acid sequences (b) and (d) are either LoxP- or LoxN nucleic acid sequences.

7. Vector system according to any one of claims 1 to 6, characterized in that it comprises a nucleic acid sequence encoding a Cre-recombinase, wherein it comprises a further nucleic acid sequence controlling the inducible expression of the Cre-recombinase.

8. A cell, characterized in that it comprises a vector system according to any one of the preceding claims.

9. The cell according to claim 8, characterized in that it is a eukaryotic cell, for example derived from a plant, an insect, a fish, a bird or a mammal.

10. A non-human multicellular organism, characterized in that it comprises a vector system according to any one of the preceding claims.

11. A Transgenic non-human multicellular organism, characterized in that it has been generated by a method comprising the following steps:

(i) Introduction of a vector system according to any one of the claims 1 to 16 into an embryo or a germ cell, (ii) Selection of hemizygous organisms expressing at least two of the transgenes which are encoded by the vector system,

(iv) Selection of descendants/offsprings which have been obtained by crossing of hemizygous organisms according to step (ii) with non-human, multicellular organisms expressing a recombinase transgene, and a subsequent out-crossing against the wildtype, and which express only one of the at least two transgenes which are encoded by the vector system according to any of the claims l to 16,

(v) Crossing of transgenic, non-human, multicellular hemizygous organisms which express only one of the at least two transgenes, with transgenic non- human, multicellular hemizygous organisms which express another of the at least two transgenes,

(viii) Identification of transgenic, non-human, multicellular homozygous descendants/offsprings, which express one the transgenes.

12. Transgenic, non-human, multicellular organism, characterized in that it has been generated by a method according to claim 22, wherein the vector system according to any of the claims 1 to 7 has been introduced into an embryo or a germ cell, which is able to express the Cre-recombinase in step (i), and wherein step (iii) of the method according to claim 11 is omitted.

13. Use of a vector system according to any of the claims 1 to 7, characterized in that it is used for the identification of hemizygous, heterozygous and/or homozygous, non- human, multicellular organisms, who are derived in a method comprising the mentioned steps of the claims 11 or 12.