Author response: A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals. https://doi.org/10.7554/eLife.31677.001 eLife digest Researchers frequently use model organisms, such as mice, zebrafish and various insect species, to understand biological processes – with the underlying idea that discoveries made can be applied to other species too. A common technique is genetic manipulation, in which a foreign gene is inserted into the chromosome of an organism. These introduced genes are called transgenes and the organisms carrying them are referred to as transgenic. Transgenic organisms are powerful tools to analyze biological processes or mimic human diseases. Many model organisms carry two homologous chromosomes – one inherited from each parent. Pairs of chromosomes carry genes in the same order, but do not necessarily have identical versions of those genes. Newly created transgenic organisms, however, carry the transgene on only one of the chromosomes. This can be a problem for researchers, as many experiments require individuals that carry the transgene on both. Unfortunately, only costly and error-prone methods can distinguish between these individuals. To overcome these drawbacks, Strobl et al. developed a concept called AGameOfClones and applied it to the red flour beetle Tribolium castaneum. In their approach, the transgene also expresses two marker-proteins with different fluorescent colors. After several generations of breeding, two versions of the transgene emerge – each retaining only one of the markers. This means that in the following generation, descendants that express both markers must be the offspring that carry the transgene on both of the chromosomes. The AGameOfClones concept has several major advantages: individuals with different markers can be easily identified, the procedure is cost-efficient and reliable, and it can be applied to nearly all model organisms. This will benefit breeding schemes and animal welfare since irrelevant individuals can be excluded as soon as the markers become detectable. https://doi.org/10.7554/eLife.31677.002 Introduction Life sciences, especially cell and developmental biology, rely on model organisms. The most frequently used vertebrates are mouse and zebrafish. Amongst insects, the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum are the two prevailing species. An important standard technique is transgenesis, that is, the insertion of recombinant DNA into the genome of the model organism (Gama Sosa et al., 2010). Since model organisms are typically diploid, the genotype has to be considered, which leads to a certain experimental complexity. Usual mating schemes result in (i) non-transgenic wild-type progeny, (ii) hemizygous transgenic progeny, that is, only the maternal or only the paternal chromosome carries the transgene, and (iii) homozygous transgenic progeny, that is, both the maternal and paternal chromosomes carry the transgene. In rare cases, the phenotype reveals the genotype, but usually, either two of the three or even all three outcomes cannot be distinguished. Transformation markers can be used to separate wild-type from transgenic, but not hemi- from homozygous individuals. Thus, additional experiments are necessary to determine the genotype, for example genetic assays, which are invasive and require manpower as well as consumables. In our AGameOfClones (AGOC) vector concept, all genotypes are directly identifiable by specifically designed distinct phenotypes, which permits the systematic creation of homozygous transgenic lines. Our approach relies on two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination results in hemizygous individuals that retain only one of the two markers and are thus phenotypically distinguishable from each other and the wild type. In the next generation, descendants that express both markers are identified as heterozygous for the transgene. Finally, a cross of two heterozygotes results in homozygous progeny that are selected by the lack of one marker. Results 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, an emerging insect model organism (Klingler, 2004; Brown et al., 2009), in conjunction with the piggyBac transposon system (Lorenzen et al., 2003; Berghammer et al., 2009), which allows semi-random genomic insertion. We developed the transformation-ready pAGOC vector (Figure 1—figure supplement 1) that contains mOrange-based (Shaner et al., 2008) and mCherry-based (Shaner et al., 2004) eye-specific (Berghammer et al., 1999) transformation markers (mO and mC, respectively). Both fluorescent proteins are spectrally separable by appropriate excitation bands and emission filters (Shaner et al., 2005). Each marker is flanked upstream by a LoxP site (Hamilton and Abremski, 1984) and downstream by a LoxN (Livet et al., 2007) site, resulting in interweaved Lox site pairs (Figure 1). Due to variations in the spacer sequences, LoxP and LoxN sites are incompatible with each other. Figure 1 with 4 supplements see all Download asset Open asset The AGameOfClones vector concept within the piggyBac-based transformation-ready pAGOC vector for Tribolium. Two fluorescence-based transformation markers, mO and mC, are embedded into a piggyBac-based transformation-ready vector, which is characterized by 3' and 5' terminal repeats (TR) necessary for genomic insertion. The markers are based on the artificial eye-specific 3×P3 promoter, the open-reading frame for the respective fluorescent protein, that is, mOrange or mCherry, and the SV40 poly(A). Each transformation marker is flanked upstream by a LoxP site (P) and downstream by a LoxN site (N), forming interweaved Lox site pairs. The markers can be detected in the eyes by using appropriate filter sets (FS). Cre-mediated recombination leads to the excision of one marker from the genome. Upon removal, the other marker remains within the genome, since the remaining LoxP and LoxN sites are incompatible. Individuals that underwent recombination give rise to progeny in which only one marker is detected in the eyes. https://doi.org/10.7554/eLife.31677.003 We injected this vector together with a piggyBac transposase-expressing helper vector (that is, pATub'piggyBac) into pre-blastoderm embryos to achieve germline transformation. All survivors, that is, F1 potential mosaics, were mated with wild types and in six of these crosses, at least one F2 (mO-mC) founder female was found among the progeny. For each cross, one founder female was mated with a wild-type male and the progeny were scored to confirm that only a single insertion had occurred (Supplementary file 1). Transgenic descendants were 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 called AGOC #1 to #6. To roughly estimate homozygous viability, two F3 (mO-mC) pre-recombination hemizygous siblings were mated and the progeny were scored (Supplementary file 2). Additionally, the insertion locations of the transgenes were determined in four of the six AGOC sublines (Supplementary file 3). Up to this step, our scheme did not differ from most standard procedures to establish transgenic lines. Systematic creation of homozygous transgenic lines The mating procedure for the systematic creation of homozygous transgenic lines (Figure 2, an comprehensive scheme is provided in Figure 2—figure supplement 1) spanned four generations and involved a transgenic helper line, ICE{HSP68'NLS-Cre} #1. This line expresses a nuclear-localized Cre recombinase (Peitz et al., 2002) under control of the heat shock protein 68b promoter (Schinko et al., 2012) and carries a mCerulean-based (Markwardt et al., 2011) eye-specific transformation marker (mCe). The procedure was performed with all six AGOC sublines and phenotypically documented for #5 and #6 (Figure 3). Figure 2 with 2 supplements see all Download asset Open asset The AGameOfClones F3 to F7 mating procedure for the systematic creation of homozygous transgenic Tribolium lines. A rounded rectangle illustrates the genotype for two independent autosomes, white bars represent the AGOC transgene location and black bars the Cre recombinase-expressing helper transgene location. A F2 (mO-mC) founder female × wild-type male cross gives rise to F3 (mO-mC) pre-recombination hemizygotes that carry mO and mC in cis configuration. A F3 (mO-mC) pre-recombination hemizygous female × mCe homozygous helper male cross results in F4 (mCe; mO-mC) double hemizygotes, in which one marker is removed through Cre-mediated recombination. Next, a F4 (mCe; mO-mC) double hemizygous female × wild-type male cross gives rise to F5 (mO) and (mC) post-recombination hemizygotes. A F5 (mO) post-recombination hemizygous female × F5 (mC) post-recombination hemizygous male sibling cross results in F6 (mO/mC) heterozygous progeny that carry mO and mC in trans configuration. Finally, a F6 (mO/mC) heterozygous female × a F6 (mO/mC) heterozygous male sibling cross gives rise to F7 (mO/mO) and (mC/mC) homozygous progeny. The percentage boxes indicate the theoretical ratio of the progeny that carry the respective genotype, the dashed line represents genotypically identical siblings. FS, filter set; rec, recombination; db, double. https://doi.org/10.7554/eLife.31677.008 Figure 3 with 2 supplements see all Download asset Open asset The AGameOfClones F3 to F7 mating procedure demonstrated for the AGOC #5 and #6 sublines. From the F3 to the F7 generation, the genotype was phenotypically determined by monitoring mCe, mO and mC. For both sublines, F7 (mO/mO) and (mC/mC) homozygotes were obtained by following the mating procedure outlined in Figure 2. The wild-type male in the second row functions as the marker control. The percentage boxes indicate the experimental (and theoretical) ratio of the progeny that displayed the respective phenotype. FS, filter set; rec, recombination; db, double. https://doi.org/10.7554/eLife.31677.011 F3 (mO-mC) pre-recombination hemizygous females, which carried mO and mC on the maternal chromosome in cis configuration, were mated with (mCe/mCe) homozygous helper males (Figure 2 and Figure 3, first row). This resulted in F4 (mCe; mO-mC) double hemizygotes in which Cre-mediated recombination occurs (Table 1, F3 row). In this hybrid generation, adults displayed a patchy expression of mO and mC within their compound eyes (Figure 3—figure supplement 1). F4 (mCe; mO-mC) double hemizygous females were mated with wild-type males (Figure 2 and Figure 3, second row). Due to Cre-mediated recombination in the germline, this resulted in F5 (mO) and (mC) post-recombination hemizygotes that carried either only mO or only mC on the maternal chromosome (Table 1, F4 row). F5 (mO) post-recombination hemizygous females were mated with F5 (mC) post-recombination hemizygous male siblings (Figure 2 and Figure 3, third row), which resulted in F6 (mO/mC) heterozygotes that carried mO on the maternal and mC on the paternal chromosome in trans configuration (Table 1, F5 row). This was demonstrated by mating F6 (mO/mC) heterozygous females with wild-type males and scoring the progeny (Table 1, F6-S row). F6 (mO/mC) heterozygous females were mated with genotypically identical male siblings (Figure 2 and Figure 3, fourth row), which resulted in F7 (mO/mO) and (mC/mC) homozygotes that carried either only mO or only mC on both, the maternal and paternal chromosome (Figure 2 and Figure 3, fifth row as well as Table 1, F6 row). This was demonstrated by mating F7 (mO/mO) and (mC/mC) homozygous females with wild-type males and scoring the progeny (Table 1, F7-O and F7-C row, respectively). Table 1 Mating procedure results for the six proof-of-principle AGOC sublines from the F3 to the F7 generation. Bold entries mark progeny that were used in the subsequent cross. F6-S, F7-O and F7-C are control crosses. No significant differences between the arithmetic means and the theoretical Mendelian ratios were found. See Source Data 1 for raw scores ordered by transgenic sublines. https://doi.org/10.7554/eLife.31677.014 GenCrossSublineProgenyTotalF3Theoretical-50.0%-----50.0%-AGOC #1-47.9% (46)-----52.1% (50)96AGOC #2-52.4% (44)-----47.6% (40)84AGOC #3-55.1% (49)-----44.9% (40)89AGOC #4-44.1% (41)-----55.9% (52)93AGOC #5-47.6% (39)-----52.4% (43)82AGOC #6-48.2% (27)-----51.8% (29)56Mean-49.2 ± 3.9%-----50.8 ± 3.9%83.3F4Theoretical25.0%25.0%12.5%12.5%12.5%12.5%---AGOC #125.6% (30)34.2% (40)4.3% (5)10.2% (12)6.0% (7)19.7% (23)--117AGOC #227.3% (36)28.9% (38)15.1% (20)8.3% (11)9.8% (13)10.6% (14)--132AGOC #325.8% (33)32.8% (42)13.3% (17)10.1% (13)5.5% (7)12.5% (16)--128AGOC #4*14.0% (14)15.0% (15)20.0% (20)13.0% (13)22.0% (22)9.0% (9)3.0% (3)4.0% (4)100AGOC #517.1% (20)40.2% (47)16.2% (19)8.5% (10)10.3% (12)7.7% (9)--117AGOC #633.6% (39)33.6% (39)8.6% (10)6.9% (8)6.1% (7)11.2% (13)--116Mean23.9 ± 7.2%30.8 ± 8.5%12.9 ± 5.6%9.5 ± 2.1%9.9 ± 6.3%11.8 ± 4.2%0.5%0.7%118.3F5Theoretical25.0%-25.0%25.0%--25.0%--AGOC #127.2% (31)-26.3% (30)23.7% (27)--22.8% (26)-114AGOC #228.9% (26)-33.3% (30)17.8% (16)--20.0% (18)-90AGOC #324.8% (30)-27.3% (33)24.8% (30)--23.1% (28)-121AGOC #419.3% (21)-22.9% (25)37.6% (41)--20.2% (22)-109AGOC #528.2% (31)-29.1% (32)12.7% (14)--30.0% (33)-110AGOC #626.2% (22)-29.7% (25)16.7% (14)--27.4% (23)-84Mean25.8 ± 3.5%-28.1 ± 3.5%22.2 ± 8.8%--23.9 ± 4.0%-104.7F6-STheoretical--50.0%50.0%----AGOC #1--46.4% (39)53.6% (44)----84AGOC #2--50.0% (49)50.0% (49)----98AGOC #3--54.0% (68)46.0% (58)----126AGOC #4--53.8% (50)46.2% (43)----93AGOC #5--51.3% (59)48.7% (56)----115AGOC #6--57.0% (49)43.0% (37)----86Mean--52.1 ± 3.7%47.9 ± 3.7%----100.3F6Theoretical--25.0%25.0%--50.0%--AGOC #1--20.3% (23)24.8% (28)--54.9% (62)-113AGOC #2--21.5% (23)35.5% (38)--43.0% (46)-117AGOC #3--22.9% (27)22.9% (27)--54.2% (64)-118AGOC #4--22.0% (29)22.7% (30)--55.3% (73)-132AGOC #5--17.5% (18)31.1% (32)--51.4% (53)-103AGOC #6--19.8% (22)24.3% (27)--55.9% (62)-111Mean--20.7 ± 1.9%26.9 ± 5.2%--52.4 ± 4.9%-115.7F7-OTheoretical--100%------AGOC #1--100% (94)-----94AGOC #2--100% (50)-----50AGOC #3--100% (49)-----49AGOC #4--100% (79)-----79AGOC #5--100% (79)-----79AGOC #6--100% (63)-----63Mean--100 ± 0%-----69.0F7-CTheoretical---100%-----AGOC #1---100% (101)----101AGOC #2---100% (105)----105AGOC #3---100% (89)----89AGOC #4---100% (54)----54AGOC #5---100% (74)----74AGOC #6---100% (64)----64Mean---100 ± 0%----81.2 *In the AGOC #4 subline, incomplete recombination occurred in the F4 (mCe; mO-mC) double hemizygous generation, as we obtained several F5 individuals that still carried both transformation markers (7.0% in total). We continued the mating procedure with the F5 (mO) and (mC) post-recombination hemizygous progeny. Table 1—source data 1 Raw scores for all mating procedure result tables ordered by transgenic sublines. https://doi.org/10.7554/eLife.31677.015 Download elife-31677-table1-data1-v2.xlsx Throughout all generations, the subline-specific scores matched the expectations. No significant differences between the respective arithmetic means and the theoretical Mendelian ratios were found. Importantly, all expected phenotypes, and thus all expected genotypes, were found in all generations and consequently, F7 (mO/mO) as well as (mC/mC) homozygotes were obtained for all six AGOC sublines. A description of the generations and their characteristics is found in Supplementary file 4. Two controls were performed with the AGOC #5 and #6 sublines to confirm proper function of the AGOC vector concept: The F3 to F7 crossing procedure was successfully conducted with (i) swapped genders (Figure 3—figure supplement 2A, Supplementary file 5) and (ii) an alternative helper subline (Figure 3—figure supplement 2B, Supplementary file 5), ICE{HSP68'NLS-Cre} #2, which carries the same Cre recombinase-expressing cassette as the #1 subline, but at a different genomic location. Systematic creation of functional homozygous AGOC lines and sublines Based on pAGOC, we developed the intermediate pAGOC{#P'#O(LA)-mEmerald} vector (Figure 1—figure supplement 2), which contains an open-reading frame for mEmerald-labeled (Shaner et al., 2005) Lifeact, a small and universal peptide tag derived from Saccharomyces cerevisiae that binds to filamentous actin (Riedl et al., 2008). With this vector, three transformation-ready derivates were created that allow expression of mEmerald-labeled Lifeact under control of either the tubulin alpha 1-like protein (Siebert et al., 2008), the zerknüllt 1 (van der Zee et al., 2005; ; Sharma et al., 2013; Panfilio et al., 2013; Hilbrant et al., 2016) or the actin-related protein 5 promoter. Additionally, we developed two more derivates that contain expression cassettes for either mEmerald-labeled beta-galactoside alpha-2,6-sialyltransferase 1 or histone H2B under control of the tubulin alpha 1-like protein promoter. With these vectors, we created five functional lines with one, three, two, three and four sublines, respectively (that is, 13 in total), which are primarily designed for fluorescence live imaging. Twelve of these sublines went through the procedure successfully, only the AGOC{ATub'H2B-mEmerald} #4 subline turned out to be heterozygous/homozygous sterile (Supplementary file 6 for the mEmerald-labeled Lifeact-expressing sublines and Supplementary file 7 for the mEmerald-labeled beta-galactoside alpha-2,6-sialyltransferase 1- and histone H2B-expressing sublines). Fluorescence live imaging of selected functional homozygous AGOC sublines We performed long-term fluorescence live imaging of the embryonic development (Strobl and Stelzer, 2016) with three of the functional (mC/mC) homozygous sublines. We used a digital scanned laser light-sheet-based fluorescence microscope (Keller et al., 2008; Keller and Stelzer, 2010) in conjunction with previously published sample preparation protocols for Tribolium (Strobl and Stelzer, 2014; Strobl et al., 2015; Strobl et al., 2017a). The AGOC{Zen1'#O(LA)-mEmerald} #2 subline allows the characterization of actin dynamics within certain extra-embryonic membrane progenitor cells during gastrulation, visualizing the actomyosin cable that closes the serosa window (Figure 4A and Video 1). The AGOC{ARP5'#O(LA)-mEmerald} #1 subline provides strong fluorescence signal in the brain and ventral nerve cord and moderate signal throughout the remaining embryonic tissue (Figure 4B and Video 2). In contrast, the AGOC{ARP5'#O(LA)-mEmerald} #2 subline provides uniform fluorescence intensity during gastrulation, germband elongation, germband retraction and dorsal closure (Figure 4C and Video 3). Figure 4 Download asset Open asset Fluorescence live imaging of selected functional (mC/mC) homozygous AGOC sublines. (A) An AGOC{Zen1'#O(LA)-mEmerald} #2 embryo during gastrulation. This subline permits the characterization of actin and actomyosin dynamics involved in serosa window closure (first row). It can also be used to describe the cytoskeleton rearrangement of the dorsal blastoderm cells (second row) and to analyze their change in appearance during differentiation to serosa cells (third row and enlarged images). (B) An AGOC{ARP5'#O(LA)-mEmerald} #1 embryo during germband retraction. In this subline, the brain and ventral nerve cord express mEmerald-labeled Lifeact on a high level, permitting the observation of neurulation. Enlarged images show the forming ganglia of the first and second thoracic segments. (C) Comparison of embryos from the AGOC{ARP5'#O(LA)-mEmerald} #1 and #2 sublines after dorsal closure. In contrast to the #2 subline, the fluorescence signal within the nervous system of the #1 subline is noticeably strong. ZP, Z maximum projection with image processing; ZA, Z maximum projection with intensity adjustment. https://doi.org/10.7554/eLife.31677.016 Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Long-term live imaging of a (mC/mC) homozygous Tribolium embryo from the AGOC{Zen1'#O(LA)-mEmerald} #2 subline. Embryogenesis is shown along four from to with an of between the The video with the rearrangement of the blastoderm and during germband retraction. gastrulation, the serosa window is by a actomyosin cable that the serosa and the is five ZP, Z maximum projection with image Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Long-term live imaging of a (mC/mC) homozygous Tribolium embryo from the AGOC{ARP5'#O(LA)-mEmerald} #1 subline. Embryogenesis is shown along four from to with an of between the The video with the rearrangement of the blastoderm and after dorsal closure. This transgenic line strong expression in the ventral nerve is five ZA, Z maximum projection with image adjustment. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Long-term live imaging of a (mC/mC) homozygous Tribolium embryo from the AGOC{ARP5'#O(LA)-mEmerald} #2 subline. Embryogenesis is shown along four from to with an of between the The video with the rearrangement of the blastoderm and after dorsal closure. In contrast to the #1 subline 2), this subline does not strong expression in the ventral nerve is five ZA, maximum projection with image adjustment. Discussion We the genetic of the AGOC vector concept and with Tribolium. The of our approach is that are in generation, since all genotypes are directly identified by specifically designed distinct can be used to systematically progeny with genotypes, as in this for the creation of homozygous lines. our concept provides many that not only to Tribolium but also to many other model (i) Our approach saves For example, to Tribolium adults with genetic assays one (Strobl et al., the same number of individuals with a microscope (ii) The concept does not require consumables. (iii) genetic assays are the of a the for our concept also of single organisms. Our approach is and thus invasive procedures are incompatible with the experimental It can be performed even of genomic DNA cannot be obtained or even the The concept simplifies transgenic organism since genotypes are determined and mating of individuals can be performed during nearly all developmental Our approach is error-prone genetic In more independent the progeny scores the phenotypically determined homozygous transgenic lines can be systematically created with by using a number of lines is only for Drosophila in the approach, the insertion location has to be our approach in and semi-random insertion Many of that occurred during our are within the Materials and methods can be identified designed distinct For example, several for the of zebrafish embryos to have et al., et al., with a in our can be used to organisms with different genotypes to their markers. The of the AGOC vector concept was with but to the of the Cre-Lox system, it work in all diploid model organisms. These various insects, zebrafish, and even plants. For many insect species, the of the vector is not It has shown that both the piggyBac transposon system and the 3×P3 promoter function in Drosophila melanogaster et al., Drosophila and and many other et al., et al., et al., species such as et al., and et al., 2010). This also to multiple such as et al., 2004) and et al., other et al., as well as for example the et al., For several other species, such as the et al., as well as several and et al., et al., 2011) and et al., et al., 2004) species, the marker cassettes have to be for example by the artificial 3×P3 promoter with the Drosophila promoter. fluorescence-based markers with the experimental markers can be markers are for Drosophila melanogaster and 2002) and Tribolium castaneum (Lorenzen et al., but require the appropriate and universal for are markers, which throughout the and thus can be detected et al., the piggyBac transposon system in zebrafish et al., and the 3×P3 promoter is to work in a of animal species (Berghammer et al., it may be to to the transposon system et al., and to the 3×P3 promoter in the marker cassettes with for example the eye-specific or the promoter and For et al., et al., 2005) and eye-specific et al., 2011) fluorescence-based as well as et al., 1999) markers have In this we used the AGOC vector concept in conjunction with transposon