Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos

Development ◽  
1988 ◽  
Vol 103 (2) ◽  
pp. 403-412 ◽  
Author(s):  
G.W. Stuart ◽  
J.V. McMurray ◽  
M. Westerfield
Keyword(s):  
Germ Line ◽  
Transmission Rate ◽  
Transgenic Fish ◽  
Early Embryo ◽  
Zebrafish Embryos ◽  
Fish Genome ◽  
Bacterial Plasmid ◽  
Foreign Dna ◽  
High Concentrations ◽  
F2 Progeny

To generate stable lines of transgenic fish, early zebrafish embryos were injected with high concentrations of a linear bacterial plasmid. After injection, the foreign DNA was converted into a high molecular weight form and then amplified approximately tenfold during the initial rapid cleavages characteristic of the early embryo prior to gastrulation. While most of this DNA was subsequently degraded during gastrulation, some of the foreign sequences survived the gastrula stage and could be found in most of the injected fish at 3 weeks of age. Only about 5% of fish analysed 4 months after the injection retained foreign DNA in their fins, usually at less than one copy per cell. One of these fish was also found to contain about 100 copies per cell of foreign DNA in a fraction of its germ cells. Approximately 20% of the F1 offspring from this germ-line-positive parent inherited the foreign DNA, whereas 50% of F2 progeny obtained from an identified F1 individual inherited these sequences. The 50% transmission rate in F2 progeny was as expected for a single, heterozygous genomic insert. These observations indicate that injected DNA can be integrated into the fish genome, that the resulting transgenic fish are mosaic and that some of these mosaic individuals give rise to stable lines of transgenic fish.

The relationship between ovarian and embryonic dorsoventral patterning in Drosophila

Development ◽  
1994 ◽  
Vol 120 (8) ◽  
pp. 2245-2257 ◽  
Author(s):  
S. Roth ◽  
T. Schupbach
Keyword(s):  
Egf Receptor ◽  
Germ Line ◽  
Early Embryo ◽  
Follicular Epithelium ◽  
Forming Process ◽  
Cell Fates ◽  
Dorsoventral Axis ◽  
Potential Ligand ◽  
Anterior Posterior ◽  
Anterior Posterior Axis

In Drosophila, the dorsoventral asymmetry of the egg chamber depends on a dorsalizing signal that emanates from the oocyte. This signal is supplied by the TGF alpha-like gurken protein whose RNA is localized to the dorsal-anterior corner of the oocyte, gurken protein is the potential ligand of the Drosophila EGF receptor homolog (torpedo), which is expressed in the follicular epithelium surrounding the oocyte. Here, we describe how changes in the dorsalizing germ-line signal affect the embryonic dorsoventral pattern. A reduction in strength of the germ-line signal as produced by mutations in gurken or torpedo does not change the slope of the embryonic dorsoventral morphogen gradient, but causes a splitting of the gradient ventrally. This leads to embryos with two partial dorsoventral axes. A change in distribution of the germ-line signal as caused by fs(1)K10, squid and orb mutations leads to a shift in the orientation of the embryonic dorsoventral axis relative to the anterior-posterior axis. In extreme cases, this results in embryos with a dorsoventral axis almost parallel to the anterior-posterior axis. These results imply that gurken, unlike other localized cytoplasmic determinants, is not directly responsible for the establishment of cell fates along a body axis, but that it restricts and orients an active axis-forming process which occurs later in the follicular epithelium or in the early embryo.

The effect of steer sera generating low or high concentrations of ammonia in culture on bovine embryo development and cell number in vitro

1999 ◽  
Vol 1999 ◽  
pp. 2-2 ◽  
Author(s):  
M. Kuran ◽  
M.E. Staines ◽  
G.J. McCallum ◽  
A.G. Onal ◽  
T.G. McEvoy
Keyword(s):  
Embryo Development ◽  
Cell Number ◽  
Early Embryo ◽  
Bovine Embryo ◽  
Cleavage Rate ◽  
Cell Monolayers ◽  
High Concentrations ◽  
Oviduct Fluid ◽  
Bovine Oocytes

Ovine embryos produced in synthetic oviduct fluid (SOF) medium or in coculture with granulosa cell monolayers supplemented with low (A; 120 μmol/l) and high (B; 190 μmol/l) ammonia-producing steer sera caused different degrees of fetal oversize (Carolan et al., 1998). The objective of the present study was to determine whether the effects on fetal growth induced by these sera were associated with alterations in early embryo development.A total of 911 bovine oocytes, used in 8 replicates to test the effect of three culture treatments on embryo development, were matured and fertilized in vitro (IVF= Day 0). Presumptive zygotes were allocated on Day 1 to culture in SOF supplemented with 10% v/v steer serum (SOF+A, n=308; SOF+B, n=302) or with amino acids plus 0.4% w/v crystalline BSA (SOFaaBSA, n=301). All cultures were in 20 μl droplets under oil (38.5°C; 5% CO2, 5% O2; 4 zygotes per drop) and droplets were renewed every 48 h. Cleavage rate was recorded on Day 3. On Days 7 and 8, blastocyst yields, grade 1 and 2 blastocysts, their cell numbers (by staining with Hoechst 33342) and their stage and diameter were determined.

scholarly journals Xist imprinting is promoted by the hemizygous (unpaired) state in the male germ line

2015 ◽  
Vol 112 (47) ◽  
pp. 14415-14422 ◽  
Author(s):  
Sha Sun ◽  
Bernhard Payer ◽  
Satoshi Namekawa ◽  
Jee Young An ◽  
William Press ◽  
...  
Keyword(s):  
X Chromosome ◽  
Germ Line ◽  
Transgenic Animals ◽  
Early Embryo ◽  
Male Meiosis ◽  
Meiotic Pairing ◽  
Male Germ Line ◽  
Specific Expression ◽  
X Chromosomes ◽  
High Level

The long noncoding X-inactivation–specific transcript (Xist gene) is responsible for mammalian X-chromosome dosage compensation between the sexes, the process by which one of the two X chromosomes is inactivated in the female soma. Xist is essential for both the random and imprinted forms of X-chromosome inactivation. In the imprinted form, Xist is paternally marked to be expressed in female embryos. To investigate the mechanism of Xist imprinting, we introduce Xist transgenes (Tg) into the male germ line. Although ectopic high-level Xist expression on autosomes can be compatible with viability, transgenic animals demonstrate reduced fitness, subfertility, defective meiotic pairing, and other germ-cell abnormalities. In the progeny, paternal-specific expression is recapitulated by the 200-kb Xist Tg. However, Xist imprinting occurs efficiently only when it is in an unpaired or unpartnered state during male meiosis. When transmitted from a hemizygous father (+/Tg), the Xist Tg demonstrates paternal-specific expression in the early embryo. When transmitted by a homozygous father (Tg/Tg), the Tg fails to show imprinted expression. Thus, Xist imprinting is directed by sequences within a 200-kb X-linked region, and the hemizygous (unpaired) state of the Xist region promotes its imprinting in the male germ line.

scholarly journals Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts

1996 ◽  
Vol 109 (6) ◽  
pp. 1555-1563 ◽  
Author(s):  
U.P. Strausfeld ◽  
M. Howell ◽  
P. Descombes ◽  
S. Chevalier ◽  
R.E. Rempel ◽  
...  
Keyword(s):  
Dna Replication ◽  
Cyclin E ◽  
Cyclin A ◽  
Early Embryo ◽  
S Phase ◽  
Chromosomal Dna ◽  
Cyclin E1 ◽  
Egg Extracts ◽  
High Concentrations ◽  
Xenopus Egg Extracts

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define ‘S-phase promoting factor’ (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

scholarly journals Locating a transgene integration site by nanopore sequencing

2019 ◽  
Author(s):  
Peter K. Nicholls ◽  
Daniel W. Bellott ◽  
Ting-Jan Cho ◽  
Tatyana Pyntikova ◽  
David C. Page
Keyword(s):  
Germ Line ◽  
Integration Site ◽  
Cost Effective ◽  
Chromosome 9 ◽  
Biological Research ◽  
Nanopore Sequencing ◽  
Fluorescent Reporter ◽  
Transgene Integration ◽  
Integration Sites ◽  
Foreign Dna

AbstractThe introduction of foreign DNA into cells and organisms has facilitated much of modern biological research, and it promises to become equally important in clinical practice. Locating sites of foreign DNA incorporation in mammalian genomes has proven burdensome, so the genomic location of most transgenes remains unknown. To address this challenge, we applied nanopore sequencing in search of the site of integration of Tg(Pou5f1-EGFP)2Mnm (also known as Oct4:EGFP), a widely used fluorescent reporter in mouse germ line research. Using this nanopore-based approach, we identified the site of Oct4:EGFP transgene integration near the telomere of Chromosome 9. This methodology simultaneously yielded an estimate of transgene copy number, provided direct evidence of transgene inversions, revealed contaminating E. coli genomic DNA within the transgene array, validated the integrity of neighboring genes, and enabled definitive genotyping. We suggest that such an approach provides a rapid, cost-effective method for identifying and analyzing transgene integration sites.

The protein product of the zebrafish homologue of the mouse T gene is expressed in nuclei of the germ ring and the notochord of the early embryo

Development ◽  
1992 ◽  
Vol 116 (4) ◽  
pp. 1021-1032 ◽  
Author(s):  
S. Schulte-Merker ◽  
R.K. Ho ◽  
B.G. Herrmann ◽  
C. Nusslein-Volhard
Keyword(s):  
Early Development ◽  
Nuclear Localization ◽  
Protein Product ◽  
Early Embryo ◽  
Paraxial Mesoderm ◽  
Zebrafish Embryos ◽  
Brachydanio Rerio ◽  
Cloning And Sequencing ◽  
Yolk Syncytial Layer ◽  
In Cells

Embryos mutant for the T gene, in mice, make insufficient mesoderm and fail to develop a notochord. We report the cloning and sequencing of the T gene in the zebrafish (Brachydanio rerio) and show the nuclear localization of the protein product. Both RNA and protein are found in cells of the germ ring, including enveloping layer cells, prior to and during gastrulation of zebrafish embryos. Nuclei of the yolk syncytial layer do not express Zf-T. High levels of expression are maintained throughout early development in the notochord, while in paraxial mesoderm cells the gene is turned off during gastrulation. Exposure of animal cap cells to activinA induces Zf-T expression, as does transplantation into the germ ring.

Multiple cis-acting targeting sequences are required for orb mRNA localization during Drosophila oogenesis

1994 ◽  
Vol 14 (4) ◽  
pp. 2235-2242
Author(s):  
V Lantz ◽  
P Schedl
Keyword(s):  
Untranslated Region ◽  
Germ Line ◽  
Critical Role ◽  
Positional Information ◽  
Mrna Localization ◽  
Early Embryo ◽  
Developmental Systems ◽  
Localization Pattern ◽  
Cis Acting ◽  
Anterior Posterior

The targeting of positional information to specific regions of the oocyte or early embryo is one of the key processes in establishing anterior-posterior and dorsal-ventral polarity. In many developmental systems, this is accomplished by localization of mRNAs. The germ line-specific Drosophila orb gene plays a critical role in defining both axes of the developing oocyte, and its mRNA is localized in a complex pattern during oogenesis. We have identified a 280-bp sequence from the orb 3' untranslated region capable of reproducing this complex localization pattern. Furthermore, we have found that multiple cis-acting elements appear to be required for proper targeting of orb mRNA.

scholarly journals The developmental effects of pentachlorophenol on zebrafish embryos during segmentation: A systematic view

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Ting Xu ◽  
Jing Zhao ◽  
Zhifa Xu ◽  
Ruijie Pan ◽  
Daqiang Yin
Keyword(s):  
Developmental Toxicity ◽  
Hypoxia Inducible Factor ◽  
Environmental Pollutants ◽  
Zebrafish Embryos ◽  
Sequencing Analysis ◽  
Developmental Effects ◽  
High Concentrations ◽  
Underlying Mechanisms ◽  
Toxicological Mechanism ◽  
Lens Formation

Abstract Pentachlorophenol (PCP) is a typical toxicant and prevailing pollutant whose toxicity has been broadly investigated. However, previous studies did not specifically investigate the underlying mechanisms of its developmental toxicity. Here, we chose zebrafish embryos as the model, exposed them to 2 different concentrations of PCP, and sequenced their entire transcriptomes at 10 and 24 hours post-fertilization (hpf). The sequencing analysis revealed that high concentrations of PCP elicited systematic responses at both time points. By combining the enrichment terms with single genes, the results were further analyzed using three categories: metabolism, transporters, and organogenesis. Hyperactive glycolysis was the most outstanding feature of the transcriptome at 10 hpf. The entire system seemed to be hypoxic, although hypoxia-inducible factor-1α (HIF1α) may have been suppressed by the upregulation of prolyl hydroxylase domain enzymes (PHDs). At 24 hpf, PCP primarily affected somitogenesis and lens formation probably resulting from the disruption of embryonic body plan at earlier stages. The proposed underlying toxicological mechanism of PCP was based on the crosstalk between each clue. Our study attempted to describe the developmental toxicity of environmental pollutants from a systematic view. Meanwhile, some features of gene expression profiling could serve as markers of human health or ecological risk.

In vivo assessment of the toxicity of electronic cigarettes to zebrafish (Danio rerio) embryos, following gestational exposure, in terms of mortality, developmental toxicity, and hair cell damage: Toxicity of E-cigs to zebrafish embryos

2020 ◽  
Vol 40 (1) ◽  
pp. 148-157
Author(s):  
YS Chang ◽  
SM Park ◽  
YC Rah ◽  
EJ Han ◽  
SI Koun ◽  
...  
Keyword(s):  
Heart Rate ◽  
Hair Cell ◽  
Hair Cells ◽  
Cell Damage ◽  
Developmental Toxicity ◽  
Zebrafish Embryos ◽  
Dorsal Fin ◽  
Gestational Exposure ◽  
Hair Cell Damage ◽  
High Concentrations

With the ban of conventional cigarettes from public spaces, electronic cigarette (E-cig) liquids have emerged as a nicotine replacement treatment for smoking cessation. However, consumers possess little knowledge of the ingredients and health effects of E-cig liquids following exposure. This study evaluated hair cell damage and developmental toxicities following gestational exposure to E-cig liquids. Zebrafish embryos were exposed to E-cig liquids at different concentrations (0.1%, 0.2%, and 0.4%). Embryonic developmental toxicity and hair cell damage was evaluated at 6 and 7 d, respectively, after fertilization. The average number of hair cells in the anterior lateral line (ALL) and posterior lateral line (PLL) following E-cig exposure was compared to that of the control. Morphological abnormalities and heart rate were evaluated. E-cig liquids significantly damaged the hair cells in the ALL, compared to the control (control; 52.85 ± 5.29 cells, 0.1% E-cig; 49.43 ± 7.70 cells, 0.2% E-cig; 40.68 ± 12.00 cells, 0.4% E-cig; 32.14 ± 20.75%; n = 29–40; p < 0.01). At high concentrations, E-cig liquids significantly damaged the hair cells in the PLL (control; 36.88 ± 5.43 cells, 0.1% E-cig; 33.06 ± 5.21 cells, 0.2% E-cig; 30.95 ± 8.03 cells, 0.4% E-cig; 23.72 ± 15.53%, n = 29–40; p < 0.01). No morphological abnormalities in body shape, somites, notochord, tail, and pectoral fin were observed; however, abnormalities were observed in the dorsal fin and heart rate at high concentrations. Thus, gestational exposure to E-cigs significantly damaged hair cells in a concentration-dependent manner and induced developmental toxicities to the dorsal fin and heart rate at high concentrations.

Impact of gonadotropins on oocyte maturation, fertilisation and developmental competence in vitro

2014 ◽  
Vol 26 (5) ◽  
pp. 752 ◽  
Author(s):  
Xuemei Wang ◽  
Tony Tsai ◽  
Jie Qiao ◽  
Zhan Zhang ◽  
Huai L. Feng
Keyword(s):  
Oocyte Maturation ◽  
Early Embryo ◽  
Developmental Competence ◽  
Dependent Manner ◽  
Success Rates ◽  
Maturation In Vitro ◽  
High Concentrations ◽  
Development In Vitro ◽  
Dose Dependent

The aim of the present study was to evaluate the dose-dependent effects of gonadotropins, either singly (Bravelle (B), Luveris (L), Menupur (M), Repronex (R), Gonal-F (G), Follism (F) and Norvarel (N)) or in combination (Menupur + Bravelle; Repronext + Bravelle; and Bravelle + Norvarel), on rates of oocyte maturation, fertilisation and early embryo development in vitro in an animal model. Bovine cumulus–oocyte complexes (COCs) were purchased commercially and cultured in TCM-199 with 10% fetal bovine serum supplemented with varying concentrations of gonadotropin (0, 5, 10, 20, 40 IU or United States Pharmacopoeia (USP) mL–1) for 24 and 48 h according to current IVF clinical stimulation protocols. All gonadotropins enhanced oocyte maturation in vitro in a dose-dependent manner. Individually, Gonal-F (Merck KGaA, Darmstadt, Germany), Follism (Merck Co, Whitehouse Station, NJ, USA) and Repronext (Ferring, Parsippany, NJ, USA) promoted oocyte maturation; in combination, they effectively enhanced COC expansion and increased the maturation competence of MII oocytes. However, high concentrations of gonadotropins may result in maturation arrest. Specific combinations of gonadotropins may change the rate of early embryonic development (8–16-cells) and morula–blastocyst formation. These data provide support for the responsiveness of bovine oocytes to gonadotropins in vitro and the need to consider variations in the relative concentrations and ratio of combinations (FSH/LH or human chorionic gonadotropin) for optimisation of oocyte developmental competence. The results of the present study could be applied to therapeutic clinical stimulation protocols and help improve IVF success rates.

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