Gene activation via Cre/lox-mediated excision in cowpea (Vigna unguiculata)

Abstract Key message Expression of Cre recombinase by AtRps5apro or AtDD45pro enabled Cre/lox-mediated recombination at an early embryonic developmental stage upon crossing, activating transgenes in the hybrid cowpea and tobacco. Abstract Genetic engineering ideally results in precise spatiotemporal control of transgene expression. To activate transgenes exclusively in a hybrid upon fertilization, we evaluated a Cre/lox-mediated gene activation system with the Cre recombinase expressed by either AtRps5a or AtDD45 promoters that showed activity in egg cells and young embryos. In crosses between Cre recombinase lines and transgenic lines harboring a lox-excision reporter cassette with ZsGreen driven by the AtUbq3 promoter after Cre/lox-mediated recombination, we observed complete excision of the lox-flanked intervening DNA sequence between the AtUbq3pro and the ZsGreen coding sequence in F1 progeny upon genotyping but no ZsGreen expression in F1 seeds or seedlings. The incapability to observe ZsGreen fluorescence was attributed to the activity of the AtUbq3pro. Strong ZsGreen expression in F1 seeds was observed after recombination when ZsGreen was driven by the AtUbq10 promoter. Using the AtDD45pro to express Cre resulted in more variation in recombination frequencies between transgenic lines and crosses. Regardless of the promoter used to regulate Cre, mosaic F1 progeny were rare, suggesting gene activation at an early embryo-developmental stage. Observation of ZsGreen-expressing tobacco embryos at the globular stage from crosses with the AtRps5aproCre lines pollinated by the AtUbq3prolox line supported the early activation mode.

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Double-inducible gene activation system for caspase 3 and 9 in epidermis

genesis ◽

10.1002/dvg.20288 ◽

2007 ◽

Vol 45 (4) ◽

pp. 194-199 ◽

Cited By ~ 2

Author(s):

Viraj R. Shah ◽

Maranke I. Koster ◽

Dennis R. Roop ◽

David M. Spencer ◽

Lei Wei ◽

...

Keyword(s):

Caspase 3 ◽

Gene Activation ◽

Activation System ◽

Inducible Gene

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Muscle gene activation in Xenopus requires intercellular communication during gastrula as well as blastula stages

Development ◽

10.1242/dev.116.supplement.137 ◽

1992 ◽

Vol 116 (Supplement) ◽

pp. 137-142 ◽

Cited By ~ 2

Author(s):

J. B. Gurdon ◽

K. Kao ◽

K. Kato ◽

N. D. Hopwood

Keyword(s):

Protein Synthesis ◽

Developmental Stage ◽

Cell Transplantation ◽

Intercellular Communication ◽

Gene Activation ◽

Gastrula Stage ◽

Blastula Stage ◽

Morphological Marker ◽

Muscle Gene ◽

Early Gastrula

In Xenopus an early morphological marker of mesodermal induction is the elongation of the mesoderm at the early gastrula stage (Symes and Smith, 1987). We show here that the elongation of equatorial (marginal) tissue is dependent on protein synthesis in a mid blastula, but has become independent of it by the late blastula stage. In animal caps induced to become mesoderm, the time when protein synthesis is required for subsequent elongation immediately follows the time of induction, and is not related to developmental stage. For elongation, intercellular communication during the blastula stage is of primary importance. Current experiments involving cell transplantation indicate a need for further celhcell interactions during gastrulation, and therefore after the vegetal-animal induction during blastula stages. These secondary cell interactions are believed to take place among cells that have already received a vegetal induction, and may facilitate some of the later intracellular events known to accompany muscle gene activation.

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A novel T-DNA vector design for selection of transgenic lines with simple transgene integration and stable transgene expression

Functional Plant Biology ◽

10.1071/fp05072 ◽

2005 ◽

Vol 32 (8) ◽

pp. 671 ◽

Cited By ~ 10

Author(s):

Song Chen ◽

Christopher A. Helliwell ◽

Li-Min Wu ◽

Elizabeth S. Dennis ◽

Narayana M. Upadhyaya ◽

...

Keyword(s):

Transgene Expression ◽

Marker Gene ◽

Direct Repeat ◽

Transgene Silencing ◽

Selective Marker ◽

Hairpin Rna ◽

Transgenic Lines ◽

Flanking Sequences ◽

Dna Vector ◽

Selection Of

Plants transformed with Agrobacterium frequently contain T-DNA concatamers with direct-repeat (d / r) or inverted-repeat (i / r) transgene integrations, and these repetitive T-DNA insertions are often associated with transgene silencing. To facilitate the selection of transgenic lines with simple T-DNA insertions, we constructed a binary vector (pSIV) based on the principle of hairpin RNA (hpRNA)-induced gene silencing. The vector is designed so that any transformed cells that contain more than one insertion per locus should generate hpRNA against the selective marker gene, leading to its silencing. These cells should, therefore, be sensitive to the selective agent and less likely to regenerate. Results from Arabidopsis and tobacco transformation showed that pSIV gave considerably fewer transgenic lines with repetitive insertions than did a conventional T-DNA vector (pCON). Furthermore, the transgene was more stably expressed in the pSIV plants than in the pCON plants. Rescue of plant DNA flanking sequences from pSIV plants was significantly more frequent than from pCON plants, suggesting that pSIV is potentially useful for T-DNA tagging. Our results revealed a perfect correlation between the presence of tail-to-tail inverted repeats and transgene silencing, supporting the view that read-through hpRNA transcript derived from i / r T-DNA insertions is a primary inducer of transgene silencing in plants.

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Sequential gene activation and gene imprinting during early embryo development in maize

The Plant Journal ◽

10.1111/tpj.13786 ◽

2018 ◽

Vol 93 (3) ◽

pp. 445-459 ◽

Cited By ~ 6

Author(s):

Dexuan Meng ◽

Jianyu Zhao ◽

Cheng Zhao ◽

Haishan Luo ◽

Mujiao Xie ◽

...

Keyword(s):

Embryo Development ◽

Gene Activation ◽

Early Embryo ◽

Early Embryo Development ◽

Gene Imprinting

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24 EFFECTS OF HISTONE METHYLATION RELATED GENES ON EPIGENETIC REPROGRAMMING AND ZYGOTIC GENE ACTIVATION IN OVINE SOMATIC CELL NUCLEAR TRANSFER (SCNT) EMBRYOS

Reproduction Fertility and Development ◽

10.1071/rdv21n1ab24 ◽

2009 ◽

Vol 21 (1) ◽

pp. 112

Author(s):

I. Choi ◽

K. H. S. Campbell

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Embryo Development ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Gene Activation ◽

Early Embryo ◽

Cell Stage ◽

Early Embryo Development ◽

Zygotic Gene

After fertilization, early embryo development is dependent upon maternally inherited proteins and protein synthesised from maternal mRNA until zygotic gene activation (ZGA) occurs. The transition of transcriptional activity from maternal to embryonic control occurs with the activation of rRNA genes and the formation of the nucleolus at the 8- to 16-cell stage that coincides with a prolonged fourth cell cycle in bovine and ovine embryos. However, previous studies have reported a shift in the longest cell cycle (fifth cell cycle) in bovine somatic cell nuclear transfer (SCNT) embryos, suggesting that the major genome activation is delayed, possibly due to incomplete changes in chromatin structure such as hypermethylation and hypoacetylation of histone (Memili and First 2000 Zygote 8, 87–96; Holm et al. 2003 Cloning Stem Cells 5, 133–142). Although global gene expression profile studies have been carried out in somatic cell nuclear transfer embryos, little is known about the expression of genes which can alter chromatin structure in early embryo development and possibly effect ZGA. To determine whether epigenetic reprogramming of donor nuclei affected ZGA and expression profiles in SCNT embryos, ZBTB33 (zinc finger and BTB domain containing 33, also known as kaiso, a methy-CpG specific repressor), BRG1(brahma-related gene 1, SWI/SNF family of the ATP-dependent chromatin remodeling complexes), JMJD1A (jumonji domain containing 1A, H3K9me2/1-specific demethylase), JMJD1C (putative H3K9-specific demethylase), and JMJD2C (H3K9me3-specific demethylase) were examined by RT-PCR at different developmental stages [germinal vesicle (GV), metaphase II (MII), 8- to 16-cell, 16- to 32-cell, and blastocyst in both parthenogenetic and SCNT embryos]. All genes were detected in parthenogenetic and SCNT blastocyts, and ZBTB33 was also expressed in all embryos at all stages tested. However, the onset of expression of JMJD1C, containing POU5F1 binding site at 5′-promoter region and BRG1 required for ZGA are delayed in SCNT embryos as compared to parthenotes (16- v. 8-cell, and blastoocyst v. 16-cell stage). Furthermore, JMJD2C containing NANOG binding sites at the 3′-flanking region was expressed in GV and MII oocytes and parthenogenetic blastocysts, whereas in SCNT embryos, JMJD2C was only observed from the 16-cell stage onwards. Interestingly, JMJD1A, which is positively regulated by POU5F1, was not detected in GV and MII oocytes but was present in blastocyst stage embryos of both groups. Taken together, these results suggest that incomplete epigenetic modifications of genomic DNA and histones lead to a delayed onset of ZGA which may affect further development and establishment of totipotency. Subsequently, aberrant expression patterns reported previously in SCNT embryos may be attributed to improper expression of histone H3K9 and H3K4 demethylase genes during early embryo development.

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CLASSIFICATION OF DROSOPHILA EMBRYONIC DEVELOPMENTAL STAGE RANGE BASED ON GENE EXPRESSION PATTERN IMAGES

Computational Systems Bioinformatics ◽

10.1142/9781860947575_0035 ◽

2006 ◽

Cited By ~ 11

Author(s):

Jieping Ye ◽

Jianhui Chen ◽

Qi Li ◽

Sudhir Kumar

Keyword(s):

Gene Expression ◽

Developmental Stage ◽

Expression Pattern ◽

Gene Expression Pattern ◽

Embryonic Developmental Stage

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Minor Splicing Factors Zrsr1 and Zrsr2 Are Essential for Early Embryo Development and 2-Cell-Like Conversion

International Journal of Molecular Sciences ◽

10.3390/ijms21114115 ◽

2020 ◽

Vol 21 (11) ◽

pp. 4115 ◽

Cited By ~ 1

Author(s):

Isabel Gómez-Redondo ◽

Priscila Ramos-Ibeas ◽

Eva Pericuesta ◽

Raúl Fernández-González ◽

Ricardo Laguna-Barraza ◽

...

Keyword(s):

Stem Cells ◽

Alternative Splicing ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Gene Activation ◽

Early Embryo ◽

Early Embryo Development ◽

Zygotic Gene ◽

Zygotic Gene Activation ◽

Induced Pluripotent

Minor splicing plays an important role in vertebrate development. Zrsr1 and Zrsr2 paralog genes have essential roles in alternative splicing, mainly participating in the recognition of minor (U12) introns. To further explore their roles during early embryo development, we produced Zrsr1mu and Zrsr2mu mutant mice, containing truncating mutations within the second zinc finger domain. Both homozygous mutant mice were viable with a normal lifespan. When we crossed a homozygous Zrsr2mu/mu female with Zrsr1mu/mu male, the double heterozygotes were non-viable, giving rise to embryos that stopped developing mainly between the 2- and 4-cell stages, just after zygotic gene activation. RNA-seq analysis of Zrsr1/2mu 2-cell embryos showed altered gene and isoform expression of thousands of genes enriched in gene ontology terms and biological pathways related to ribosome, RNA transport, spliceosome, and essential zygotic gene activation steps. Alternative splicing was analyzed, showing a significant increase in intron retention in both U2 and U12 intron-containing genes related to cell cycle and mitotic nuclear division. Remarkably, both Zrsr1 and Zrsr2 were required for the conversion of mouse-induced pluripotent stem cells into 2C-like cells. According to our results, Zrsr1 or Zrsr2 are necessary for ZGA and both are indispensable for the conversion of induced pluripotent stem cells into 2C-like cells.

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Inducible control of tissue-specific transgene expression in Xenopus tropicalis transgenic lines

Mechanisms of Development ◽

10.1016/s0925-4773(02)00219-8 ◽

2002 ◽

Vol 117 (1-2) ◽

pp. 235-241 ◽

Cited By ~ 39

Author(s):

Jeiwook Chae ◽

Lyle B. Zimmerman ◽

Robert M. Grainger

Keyword(s):

Transgene Expression ◽

Xenopus Tropicalis ◽

Tissue Specific ◽

Transgenic Lines

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Embryogeny of Phaseolus coccineus: the ultrastructure and development of the suspensor

Canadian Journal of Botany ◽

10.1139/b79-021 ◽

1979 ◽

Vol 57 (2) ◽

pp. 120-136 ◽

Cited By ~ 46

Author(s):

Edward C. Yeung ◽

Mary E. Clutter

Keyword(s):

Smooth Endoplasmic Reticulum ◽

Early Embryo ◽

Wall Thickening ◽

Ontogenetic Changes ◽

Early Embryo Development ◽

Wall Ingrowths ◽

Globular Stage ◽

Cotyledon Stage ◽

Suspensor Cells ◽

Cellular Organelles

Structural specializations occur in the suspensor at the proembryo stage with the formation of wall ingrowths. Soon after the appearance of the ingrowths, mitochondria, plastids, polysomes, and smooth endoplasmic reticulum greatly increase in their abundance within the suspensor cells. By the late globular stage, wall ingrowths are extremely well developed and the cells within the suspensor are packed with cellular organelles. The nucleus of the suspensor cell at the heart stage also takes on an active appearance. The nucleus becomes highly lobed in shape and micronucleoli can also be found. There is little change in the overall ultrastructural pattern of the cell till the cotyledon stage. At this stage, wall thickening occurs in all suspensor cells and the outlines of most of the ingrowths become less obvious. The mitochondria, plastids, and polysomes become less abundant and a change in the configuration of the smooth endospermic reticulum is also observed. The ontogenetic changes of the cellular organelles suggest that the suspensor plays a role during early embryo development, i.e., globular to early cotyledon stage.

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