Study On Safe And High Efficient Transgenic Technology Of Silkworm, Bombyx Mori

Transgenic technology is an important tool for gene function analysis and genetic improvement of animals and plants. Since it was established in the early 1970s and after nearly half a century of development, the transgenic technology has become more and more mature. In recent years, transgenic technology has gradually been widely applied to achieve the identification and functional analysis of genes, directional genetic breeding, establishment of disease models and bioreactor. But transgenic technology since the date of birth, the scientific community and the public have long been concerned about the safety of genetically modified organisms (GMOs), the ongoing societal debate on the safety of GMOs has never stopped. At present, the safety problems of GMOs mainly include the safety of genetic manipulation techniques and the safety of genetically modified products (GMPs). The safety problems of genetic manipulation techniques are mainly reflected in whether the insertion of the exogenous genes into the genome of plants and animals that would reduced fitness of the transgenic strain and destroyed the environment. The safety problems of GMPs are mainly reflected in whether the genetically modified foods (mainly refers to the crops) and the antibodies, vaccines and other pharmaceutical products from bioreactor, or other biological products from other genetic engineering technologies, which will cause potential harm to human health. Therefore, how to utilize and combine many different genomic manipulation techniques, and solve the potential safety hazard of GMOs, thus eliminating the fears of the safety of GMOs from the public, was the key problem for the GMOs breeding, development and application which must be solved.The silkworm, Bombyx mori, is a valuable economic insect that has been do mesticated and reared for silk production for more than 5000 years. Since the first report of germline transformation with the piggyBac transposon in silkworm, the transgenic technology has been widely applied in the silkworm gene function identification, bioreactor developed and creation of new silkworm varieties with specific genetic differences. The piggyBac transposon was recognized as the most effective gene vector to established transgenic silkworm at present, but safety problems still exist due to the random integration of the transposon and the postinsertion instability of the transgene mediated by piggyBac, as well as selectable marker genes remain in the genome and other defects. The safety problems of transgenic silkworm are mainly reflected in the safety of genetic manipulation techniques, which mainly consists of the following questions:(1) Selectable marker gene sequences and the backbone sequences of piggyBac transposon and any other non-target gene sequences were remain in the genome of the transgenic silkworm, which would cause potential safety hazard. (2) Position effects and insertional mutagenesis during piggyBac-mediated random integration can have several side effects, including unpredictable variations in gene expression and the reduced fitness of the transgenic strain. (3) The phenomenon of postinsertion instability of the transgene caused by piggyBac transposon remobilization, which may result in the loss of transgene in the offspring of the transgenic silkworm or transgene escape to other species via horizontal gene transfer, and thus damage the environment. In addition, the transgenic silkworm that obtained using piggy Bac-mediated germline which will also affect gene function research, including the selection marker genes or the vector backbone sequences were present in the genome that may affect the transgene expression, and it is also almost impossible to repeatedly introduce several different exogenous genes into a specific target locus using only piggyBac transposon. In recent years, the safety problems of transgenic silkworm have become the key problems that hindering the industrialization development of transgenic silkworm. The present research on the safety problems of transgenic silkworm was not in-depth, so the above problems have yet to be resolved.In order to effectively overcome these safety problems of transgenic silkworm discussed above, this study first describe a method using the FLP/FRT site-specific recombination system to achieve site-specific excision of a target transgene at a predefined chromosomal site in transgenic silkworm genome. This mathod can be used to achieve high-efficiency and inducible site-specific excision of selective marker genes from transgenic silkworm genome, thus effectively overcome the potential safety hazard caused by the selective marker genes remain. Next, this study developed a phiC31/att site-specific recombination system to produce site-specific transgene integration into predetermined genomic sites in the transgenic silkworm genome via phiC31 integrase-mediated recombinase-mediated cassette exchange (phiC31-RMCE) reaction. This mathod can be used to introduce several different exogenous genes into a specific target locus of the transgenic silkworm, thus effectively minimize the position effect transgene expression that results from transposon-mediated random insertions into the silkworm genome. Finally, this study used a combination of site-specific recombination system and a composite piggyBac-derived vector to develop a generic and efficient strategy for stability of transgene integration and expression, and replaceable of trangene in the silkworm. Furthermore, we have established a stable, replaceable, and highly efficient transgene expression system in the silkworm based on this strategy. The results are as follows:1. In vivo site-specific excision of transgene in silkwormFirst, we established one FLP/FRT system transgenic target strain (TTS) via embryo microinjection-based germline transformation method. The molecular identification results showed that the TTS silkworms contained a single copy of the FRT-flanked A3-EGFP expression cassette in their genomes. The FLP mRNA or a FLP recombinase helper expression vector pSLA3-FLP as a source of FLP recombinase was introduced into the TTS silkworms using pre-blastoderm microinjection. Finally, the recombinase-mediated site-specific recombination strain (SSRS) silkworms without A3-EGFP expression cassette in their genomes were successful identified from the TTS offspring. The results showed that the percentage of the positive broods containing SSRS silkworms in the broods from TTS silkworms using FLP mRNA as a source of FLP recombinase (13.3%) was obvious higher than that using pSLA3-FLP vector as a source (2.38%).Next, we established one FLP recombinase-expressing helper strain (H-A-1) containing the silkwrom cytoplasmic actin 3 promoter-driving FLP expression cassette (A3-FLP) and one FLP recombinase-expressing helper strain (H-H-1) containing the Drosophila hsp70 promoter-driving FLP expression cassette (Hsp70-FLP) also via embryo microinjection-based germline transformation method, respectively. We expect that the TTS silkworms could backcrossed with the H-A-1 silkworms to produce TTS&H-A-1 double-transgenic silkworms, thus introduce the expression of FLP recombinase and achieve the high-efficient excision of the A3-EGFP expression cassette in the TTS&H-A-1 offspring. In addition, we also expect that the TTS silkworms could backcrossed with the H-H-1 silkworms to produce TTS&H-H-1 double-transgenic silkworms and by 42℃ heat shock treatment (HST), thus introduce the expression of FLP recombinase and achieve the induced excision of the A3-EGFP expression cassette in the TTS&H-H-1 offspring. To ensure the expression of FLP recombinase gene in H-A-1 and H-H-1 individuals, RT-PCR and qRT-PCR were performed. The results showed that FLP recombinase was highly expressed in larval fat body, midgut, silk gland, testis and ovary tissues and embryos of H-A-1 individuals as expected. The expression of FLP recombinase in all larval tissues and embryos of H-H-l individuals exposed to 42℃ treatment were up-regulated compared to the H-H-1 individuals without heat shock treatment, and the expression of FLP recombnase in the testis, ovary and embryos of H-H-1 individuals were significantly up-regulated 58.2%-72.3%.The results of statistical analysis showed that the frequencies of positive broods containing at least one SSRS individual in the broods from TTS&H-A-1 silkworms were reached 97.01%～100% by sexual hybridization method. The frequency of site-specific gene excision in offspring of TTS&H-H-1 silkworms by heat shock treatment (32.14%～36.67%) was significantly higher than that without heat shock treatment (2.63%-6.67%). Then molecular identification were performed on genomic DNAs from TTS silkworms and SSRS silkworms, and the results also verified that the precise site-specific recombination between two FRT sites in the genome of these TTS individuals was mediated by FLP recombinase.The strategies and methods discribed above can be used to remove selective marker genes with high-efficiency and induced from transgenic silkworm genome and thus effectively eliminate the potential biological safety problems and negative effects of selective marker genes for gene function research in transgenic silkworm.2. In vivo site-specific integration of transgene in silkwormFirst, we established two transgenic target strains TTSl-P and TTS1-B via embryo microinjection-based germline transformation method. The molecular identification results showed TTS1-P and TTS1-B contained a single copy of the predetermined genomic target site(attP/attP or attB/attB site) in their genomes, and the piggyBac inserts in the genome of TTS1-P and TTS1-B were located on chromosomes 20 (autosome) and 1 (Z chromosome), respectively. TTS1-P and TTS1-B males were backcrossed with wild-type Dazao females and sibling-mated for several generations to create TTS8-P and TTS8-B. Finally, the silkworms TTS8-P (++A♀ and++A♂, "A" indicates the transgene, as below) and TTS8-B (ZAW♀ and ZZA♂) containing attP/attP or attB/attB at homozygous or hemizygous target sites were established, respectively.The frequencies of positive broods (SSIS1-P or SSIS1-B) containing at least one site-specific integration strain (SSIS) individual from TTS10 broods were 3.84%～7.01% after co-injection of a phiC31 helper vector or phiC31 integrase mRNA with a donor vector that cotaining an attB- or attP-flanked 3×P3-EGFP expression cassette (pSL{attB1-3×P3-EGFP-SV40-attB2} or pSL{attP1-3×P3-EGFP-SV40-attP2}) into pre-blastoderm embryos of TTS9-P or TTS9-B. The molecular identification results suggest integration of the 3×P3-EGFP expression cassette by RMCE, rather than integration of the entire donor vector by a single attB/attP pair recombination, in all the selected integration strains. The results suggest that RMCE events between chromosomal attP/attP target sites and incoming attB/attB sites were more frequent than those in the reciprocal direction. Fluorescence and molecular analysis confirmed that site-specific integration of the transgene was stable in SSIS offspring.The current study demonstrated a method to achieve precise integration of exogenous DNA at pre-defined chromosomal target sites in the transgenic silkworm genome via phiC31-mediated RMCE, which overcomes the disadvantages of the position effects for transgene expression and functional genomic research incurred by transposon-mediated random integration.3. Establishment of a stable, replaceable, safe and highly efficient transgenic technique in silkwormFirst, a composite piggyBac-derived vector PB-TP containing four piggyBac arms (L1, L2, R1 and R2), a piggyBac transposase gene expression cassette (Hsp70-PBase-SV40), the selective marker genes (3×P3-DsRed and 3×P3-EGFP) and the attP-flanked FibH-EGFP-LBS expression cassette sequences was constructed. The PB-TP vector was injected into the GO B. mori 871 strain non-diapause embryos with a transposase expression helper vector pHA3PIG at the pre-blastoderm stage. Four G1 transgenic individuals with three different fluorescent phenotypes (3×P3-DsRed, R; FibH-EGFP, g; 3×P3-EGFP, G) were obtained from four different positive G1 broods for subsequent experiments and designated TS1-RgG1-TS1-RgG4. The results of molecular analysis showed that both the TS1-RgG1 and TS1-RgG2 silkworms contained only one copy of the transgene construct with four complete piggyBac arms and the attP-flanked FibH-EGFP-LBS expression cassette sequence in their genomes, and the content of pure EGFP in the TS1-RgG2 silkworm cocoon was up to 16% (w/w).One heterozygous TS1-RgG2 adult was backcrossed with a wild-type 871 adult to produce G2 broods. The G2 eggs from each G2 brood were divided into three groups (1#,2#, and 3#), the individuals in different groups from the same G2 brood were treated with HST in the embryonic stage (group 2#), in the larval stage (group 3#), or without HST (group 1#). The TS2-RgG2 transgenic individuals were screened from these G2 individuals of each group. TS2-RgG2 fertile adults were backcrossed with adults from the 871 strain to produce G3 broods, and the fluorescence phenotypes of the larvae from these G3 broods of each group were analyzed. The frequencies of positive broods containing at least one TS3-g2 silkworm (designated g-positive brood) in the G3 broods of groups 2# and 3# were 16.22% (6/37) and 2.22%(1/45), respectively, and there was no g-positive brood in any of the G3 broods from group 1#. The results of molecular analysis showed that only the complete attP-flanked FibH-EGFP-LBS expression cassette without any transposon element was retained in the genomes of the TS3-g2 individuals, and there was no footprint or any structural changes, such as deletions, inversions, or rearrangements, at the excision-site TTAA elements or attP sites in the genomes of the TS3-g2 individuals.To identify the optimal HST strategy for producing transposon-free transgenic silkworms, one heterozygous TS3-gG2 male was backcrossed with three different 871 females to produce three G4 broods. The G4 eggs from each G4 brood were divided into three groups, and the G4 individuals from the three groups of each G4 brood were treated with or without HST, as described above. Finally, the fluorescent phenotypes of the larvae from G5 broods of each group were analyzed. The results suggest that the frequency of g-positive broods (containing at least one TS5-g2 silkworm) in the G5 broods of group 1# (without HST), group 2# (HST in the embryonic stage), and group 3# (HST in the larval stage) were 0%-2%,70%-80%, and 10%-14%, respectively. All these results suggest that continuous and repeated HST at 42℃ in the embryonic stage of transgenic silkworms is the most effective way to delete the transposons from their offspring.The heterozygous TS4-g2 (+/-, indicates a heterozygote), homozygous TS4-g2 (+/+, indicates a homozygote) and heterozygous TS4-gG2 (+/-) adults were selected and backcrossed with 871 adults to produce G5 broods, respectively, and the fluorescent phenotypes of the larvae from these G5 broods were analyzed. The results showed that the rate of TS5-g2 individuals from each of the G5 broods obtained by the reciprocal crosses between TS4-g2 (+/-) adults and 871 adults were almost exactly 50% (if the g is stable, the theoretical rate of g phenotype in these G5 broods should be 50%), the rate of TS5-g2 individuals from each of the G5 broods obtained by the reciprocal crosses between TS4-g2 (+/+) adults and 871 adults were all 100% (if the g is stable, the theoretical rate of g phenotype in these G5 broods should be 100%), and the rates of G5 individuals with g fluorescence phenotype (including TS5-gG2 and TS5-g2 individuals) from each of the G5 broods obtained by the TS4-gG2 (+/-) adults backcrossed with 871 adults were also almost exactly 50%, which was consistent with the theoretical value. In addition, EGFP expression was detected in TS7-g2 individuals and their cocoons. The results showed that EGFP was highly efficiently and specifically expressed in the silk glands of the TS7-g2 silkworms and was spun into their cocoons. The PCR analysis confirmed that the FibH-EGFP expression cassette was stable in TS3-g2 offspring. All the above results confirmed that the integration of FibH-EGFP expression cassette and the expression of EGFP were stable in the TS3 offspring whether PBase is present or absence in vivo.First, this study used piggyBac-mediated germline transformation to generate a G1 transgenic silkworm strain that produces exogenous proteins with high efficiency in the silk gland and characterized the strain. The subsequent elimination of all transposon sequences containing the PBase gene expression cassette and all the marker genes for G2 transgenic silkworm was completed with the heat-shock-induced expression of the transposase in vivo, generating G3 transgenic silkworm, which contains only the attP-flanked optimized fibroin H-chain expression cassette in its genome. This method not only prevents the remobilization of the target gene, but also eliminates the adverse effects of the selectable marker genes and the backbone sequences of transposon in any future application of the transgenic insect, which may affect the expression of the target genes, the growth and development of the transgenic individuals, horizontal gene transfer, or any of the other potential ecological security problems associated with transgenic insects. A phiC31-RMCE method could be used to integrate other genes of interest into the same genome locus between the attP sites in G3 transgenic silkworm. Controlling for position effects with phiC31-RMCE will also allow the optimization of exogenous protein expression and fine gene function analyses in the silkworm. The strategy developed here is also applicable to other lepidopteran insects, to improve the ecological safety of transgenic strains in biocontrol programs.