A <i>piggyBac</i> transposon‐ and gateway‐enhanced system for efficient BAC transgenesis

Zhao, Liang; Ng, Ee Ting; Koopman, Peter

doi:10.1002/dvdy.24153

Cited by 19 publications

(15 citation statements)

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“…Using the piggyBac-enabled Wt1 -BAC system [18] (Fig. 1A), we successfully generated two transgenic mouse lines (Tg-A and Tg-S) expressing an Nr5a1- IRES- Egfp transgene at different levels (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The mouse transgenesis procedure was based on a protocol described previously [18]. Briefly, a sequence containing the mouse Nr5a1 coding region followed by an internal ribosomal entry site and the sequence encoding enhanced green fluorescent protein ( Nr5a1 -IRES- Egfp ) was cloned into the PBWt1-Dest vector via Gateway LR recombination (Fig.…”

Section: Methodsmentioning

confidence: 99%

“…To address these questions, we exploited a BAC (bacterial artificial chromosome) transgene system [18] whereby Nr5a1 expression is driven by Wt1 regulatory sequences. In the fetal XX gonads, endogenous Wt1 is expressed in supporting cells including pre-granulosa cells and coelomic epithelium [19-21].…”

Section: Introductionmentioning

confidence: 99%

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Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

Nomura

Kashimada

Suzuki

et al. 2018

Preprint

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26The nuclear receptor NR5A1 (also known as Ad4BP, or SF1) is essential for the initial steps 27 of mammalian gonadal development. The Nr5a1 gene is equally expressed in XX and XY 28 gonadal primordia, but after sex determination, is up-regulated in XY and down-regulated in 29 XX gonads. We recently reported a case of 46, XX disorder of sex development (DSD) in 30 which ectopically expressed NR5A1 in XX gonads led to an ovo-testicular phenotype, 31 suggesting that excess NR5A1 can direct the development of immature XX gonads towards 32 testicular formation. However, a direct causal relationship has not been demonstrated in an 33 animal model. Here, using a Wt1-BAC (bacterial artificial chromosome) transgene system, 34 we generated two lines of mice overexpressing Nr5a1 in the fetal gonads at different levels.35 One of these lines (Tg-S), highly expressing Nr5a1, revealed that enforced Nr5a1 expression 36 alone is insufficient to switch the fate of the 46,XX gonads toward testicular formation in 37 mice. In the other line (Tg-A) expressing Nr5a1 at lower level, ovarian development was 38 compromised, with multi-oocyte follicles, reduced number of matured follicles, and impaired 39 expression of Wnt4, resulting in late onset infertility at 20 weeks after birth. The phenotype 40 was similar to that of genetically modified mice with impaired Notch signaling. Indeed, the 41 expression level of Notch2 and 3 was significantly reduced in Tg-A mice, and the ovarian 42 phenotype in Tg-A mice was almost completely rescued by in utero treatment with a Notch2 43 agonist HMN2-29. We conclude that suppression of Nr5a1 during the fetal period optimizes 44 ovarian development by fine tuning of Notch signaling levels. 45( 257 words) 46 47 AUTHOR SUMMARY 48 Sexual development is a process of differentiation from undifferentiated bipotential gonads, 3 50 understanding of organogenesis. The nuclear receptor NR5A1 which is essential for 51 mammalian gonadal development, is equally expressed in both gonadal primordia, but after 52 sex determination, is up-regulated in XY and down-regulated in XX gonads. We have 53 recently demonstrated that this down-regulation is mediated by ovarian transcription factor, 54 Forkhead box L2 (FOXL2). This finding raised two key questions, whether Nr5a1 can 55 function as a male sex-determining factor, and whether the repression is essential for 56 appropriate ovarian development. By generating two lines Tg mice in XX gonads with 57 different enforced expression levels of Nr5a1, our present study revealed that alterations in 58 Nr5a1 dosage, either reduced or excessive, result in pathological effects in ovarian 59 development and female fertility, indicating that the precise control of Nr5a1 at the 60 transcriptional level is essential for optimal ovarian development. We envisage that the 61 improved understanding of how this pathway regulates ovarian development and female 62 fertility would aid the development of artificial somatic ovarian cells, which in turn may 63 provide a valuable treatment option...

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

Nomura

Kashimada

Suzuki

et al. 2018

Preprint

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“…The herein demonstrated normal growth and reproduction performance, harmless genomic insertion, stable transmission and expression of transgene in transgenic pigs generated with the PB transposase, together with the ability of the PB, Tol2 and SB transposases in integrating large size of bacterial artificial chromosome (BAC) constructs into TAs genomes [28–30], makes the transpositional “active transgenesis” an exciting method for production of TAs.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

Zeng

GengYuan

et al. 2016

Animal Biotechnology

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Previously we successfully produced a group of EGFP-expressing founder transgenic pigs by a newly developed efficient and simple pig transgenesis method based on cytoplasmic injection of piggyBac plasmids. In this study, we investigated the growth and reproduction performance, and characterized the transgene insertion, transmission and expression patterns in transgenic pigs generated by piggyBac transposition. Results showed that transgene has no injurious effect on the growth and reproduction of transgenic pigs. Multiple copies of monogenic EGFP transgene were inserted at noncoding sequences of host genome, and passed from founder transgenic pigs to their transgenic offspring in segregation or linkage manner. The EGFP transgene was ubiquitously expressed in transgenic pigs, and its expression intensity was associated with transgene copy number but not related to its promoter DNA methylation level. To the best of our knowledge, this is first study that fully described the growth and reproduction performance, transgene insertion, expression and transmission profiles in transgenic pigs produced by piggyBac system. It not only demonstrates that piggyBac transposition-mediated gene transfer is an effective and favourable approach for pig transgenesis, but also provides scientific information for understanding the transgene insertion, expression and transmission patterns in transgenic animals produced by piggyBac transposition.

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“…To address these questions, we exploited a bacterial artificial chromosome (BAC) transgene system (Zhao et al, 2014), whereby Nr5a1 expression is driven by Wt1 regulatory sequences. In the fetal XX gonads, endogenous Wt1 is continuously expressed in supporting cells including pre-granulosa cells and coelomic epithelium (Bandiera et al, 2015;Maatouk et al, 2012;Rackley et al, 1993).…”

Section: Introductionmentioning

confidence: 99%

Nr5a1 suppression during the murine fetal period optimizes ovarian development by fine-tuning Notch signaling

Nomura

Kashimada

Suzuki

et al. 2019

Journal of Cell Science

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The nuclear receptor NR5A1 is equally expressed and required for development of the gonadal primordia of both sexes, but, after sex determination, it is upregulated in XY testes and downregulated in XX ovaries. We have recently demonstrated, in mice, that this downregulation is mediated by forkhead box L2 (FOXL2) and hypothesized that adequate suppression of Nr5a1 is essential for normal ovarian development. Further, analysis of human patients with disorders/differences of sex development suggests that overexpression of NR5A1 can result in XX (ovo)testicular development. Here, we tested the role of Nr5a1 by overexpression in fetal gonads using a Wt1-BAC (bacterial artificial chromosome) transgene system. Enforced Nr5a1 expression compromised ovarian development in 46,XX mice, resulting in late-onset infertility, but did not induce (ovo)testis differentiation. The phenotype was similar to that of XX mice lacking Notch signaling. The expression level of Notch2 was significantly reduced in Nr5a1 transgenic mice, and the ovarian phenotype was almost completely rescued by in utero treatment with a NOTCH2 agonist. We conclude that suppression of Nr5a1 during the fetal period optimizes ovarian development by finetuning Notch signaling.

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A piggyBac transposon‐ and gateway‐enhanced system for efficient BAC transgenesis

Cited by 19 publications

References 50 publications

Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

Nr5a1 suppression during the fetal period optimizes ovarian development by fine-tuning of Notch signaling

Characterization of Growth and Reproduction Performance, Transgene Integration, Expression, and Transmission Patterns in Transgenic Pigs Produced by piggyBac Transposition-Mediated Gene Transfer

Nr5a1 suppression during the murine fetal period optimizes ovarian development by fine-tuning Notch signaling

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