scholarly journals Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice

2021 ◽  
Author(s):  
Rebekka Medert ◽  
Thomas Thumberger ◽  
Tinatini Tavhelidse ◽  
Tobias Hub ◽  
Tanja Kellner ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Mouse Genome ◽  
Single Copy ◽  
Mouse Embryos ◽  
Large Fragment ◽  
Cell Stage ◽  
Homology Directed Repair ◽  
Independent Gene

ABSTRACTCRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.

Human Adenovirus Uptake by Preirnplantation Mouse Embryos

1972 ◽  
Vol 30 ◽  
pp. 268-269
Author(s):  
D. G. Chase ◽  
W. Winters ◽  
L. Piko
Keyword(s):  
Hela Cells ◽  
Human Adenovirus ◽  
Mouse Embryos ◽  
Equilibrium Density ◽  
Cell Stage ◽  
Virus Attachment ◽  
Preimplantation Mouse Embryos ◽  
Embryo Culture Medium ◽  
Preimplantation Mouse ◽  
Mouse Cells

Although the outlines of human adenovirus entry and uncoating in HeLa cells has been clarified in recent electron microscope studies, several details remain unclear or controversial. Furthermore, morphological features of early interactions of human adenovirus with non-permissive mouse cells have not been extensively documented. In the course of studies on the effects of human adenoviruses type 5 (AD-5) and type 12 on cultured preimplantation mouse embryos we have examined virus attachment, entry and uncoating. Here we present the ultrastructural findings for AD-5.AD-5 was grown in HeLa cells and purified by successive velocity gradient and equilibrium density gradient centrifugations in CsCl. After dialysis against PBS, virus was sedimented and resuspended in embryo culture medium. Embryos were placed in culture at the 2-cell stage in Brinster's medium.

scholarly journals Totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Marino Maemura ◽  
Hiroaki Taketsuru ◽  
Yuki Nakajima ◽  
Ruiqi Shao ◽  
Ayaka Kakihara ◽  
...  
Keyword(s):  
Inner Cell Mass ◽  
Cell Mass ◽  
Mouse Embryos ◽  
Primitive Endoderm ◽  
Cell Stage ◽  
Supportive Environment ◽  
Mouse Zygotes ◽  
Single Blastomeres ◽  
Multicellular Organisms

AbstractIn multicellular organisms, oocytes and sperm undergo fusion during fertilization and the resulting zygote gives rise to a new individual. The ability of zygotes to produce a fully formed individual from a single cell when placed in a supportive environment is known as totipotency. Given that totipotent cells are the source of all multicellular organisms, a better understanding of totipotency may have a wide-ranging impact on biology. The precise delineation of totipotent cells in mammals has remained elusive, however, although zygotes and single blastomeres of embryos at the two-cell stage have been thought to be the only totipotent cells in mice. We now show that a single blastomere of two- or four-cell mouse embryos can give rise to a fertile adult when placed in a uterus, even though blastomere isolation disturbs the transcriptome of derived embryos. Single blastomeres isolated from embryos at the eight-cell or morula stages and cultured in vitro manifested pronounced defects in the formation of epiblast and primitive endoderm by the inner cell mass and in the development of blastocysts, respectively. Our results thus indicate that totipotency of mouse zygotes extends to single blastomeres of embryos at the four-cell stage.

scholarly journals STAT3 Is an Upstream Regulator of Granzyme G in the Maternal-To-Zygotic Transition of Mouse Embryos

2021 ◽  
Vol 22 (1) ◽  
pp. 460
Author(s):  
Huan Ou-Yang ◽  
Shinn-Chih Wu ◽  
Li-Ying Sung ◽  
Shiao-Hsuan Yang ◽  
Shang-Hsun Yang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Fluorescent Protein ◽  
Mouse Embryos ◽  
Cell Nuclei ◽  
Cell Stage ◽  
Promoter Assay ◽  
Maternal To Zygotic Transition ◽  
Mouse Zygotes ◽  
Time Specific

The maternal-to-zygotic transition (MZT), which controls maternal signaling to synthesize zygotic gene products, promotes the preimplantation development of mouse zygotes to the two-cell stage. Our previous study reported that mouse granzyme g (Gzmg), a serine-type protease, is required for the MZT. In this study, we further identified the maternal factors that regulate the Gzmg promoter activity in the zygote to the two-cell stage of mouse embryos. A full-length Gzmg promoter from mouse genomic DNA, FL-pGzmg (−1696~+28 nt), was cloned, and four deletion constructs of this Gzmg promoter, Δ1-pGzmg (−1369~+28 nt), Δ2-pGzmg (−939~+28 nt), Δ3-pGzmg (−711~+28 nt) and Δ4-pGzmg (−417~+28 nt), were subsequently generated. Different-sized Gzmg promoters were used to perform promoter assays of mouse zygotes and two-cell stage embryos. The results showed that Δ4-pGzmg promoted the highest expression level of the enhanced green fluorescent protein (EGFP) reporter in the zygotes and two-cell embryos. The data suggested that time-specific transcription factors upregulated Gzmg by binding cis-elements in the −417~+28-nt Gzmg promoter region. According to the results of the promoter assay, the transcription factor binding sites were predicted and analyzed with the JASPAR database, and two transcription factors, signal transducer and activator of transcription 3 (STAT3) and GA-binding protein alpha (GABPα), were identified. Furthermore, STAT3 and GABPα are expressed and located in zygote pronuclei and two-cell nuclei were confirmed by immunofluorescence staining; however, only STAT3 was recruited to the mouse zygote pronuclei and two-cell nuclei injected with the Δ4-pGzmg reporter construct. These data indicated that STAT3 is a maternal transcription factor and may upregulate Gzmg to promote the MZT. Furthermore, treatment with a STAT3 inhibitor, S3I-201, caused mouse embryonic arrest at the zygote and two-cell stages. These results suggest that STAT3, a maternal protein, is a critical transcription factor and regulates Gzmg transcription activity in preimplantation mouse embryos. It plays an important role in the maternal-to-zygotic transition during early embryonic development.

Enhanced development of 8-cell stage blastomeres by intact mouse embryos

1992 ◽  
Vol 37 (1) ◽  
pp. 246
Author(s):  
L.Y. Li ◽  
R.S. Denniston ◽  
W. hansel ◽  
R.A. Godke
Keyword(s):  
Mouse Embryos ◽  
Cell Stage ◽  
Intact Mouse

scholarly journals Gene targeting facilitated by engineered sequence-specific nucleases and its potential for applications in crop improvement

2021 ◽  
Author(s):  
Daisuke Miki ◽  
Rui Wang ◽  
Jing Li ◽  
Dali Kong ◽  
Lei Zhang ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Agronomic Traits ◽  
Higher Plants ◽  
Crop Improvement ◽  
Strand Break ◽  
End Joining ◽  
Homology Directed Repair ◽  
Target Dna ◽  
Targeted Gene Editing ◽  
Non Homologous End Joining

Abstract Humans are currently facing the problem of how to ensure that there is enough food to feed all of the world’s population. Ensuring that the food supply is sufficient will likely require the modification of crop genomes to improve their agronomic traits. The development of engineered sequence-specific nucleases (SSNs) paved the way for targeted gene editing in organisms, including plants. SSNs generate a double-strand break (DSB) at the target DNA site in a sequence-specific manner. These DSBs are predominantly repaired via error-prone non-homologous end joining (NHEJ), and are only rarely repaired via error-free homology-directed repair (HDR) if an appropriate donor template is provided. Gene targeting (GT), i.e., the integration or replacement of a particular sequence, can be achieved with combinations of SSNs and repair donor templates. Although its efficiency is extremely low, GT has been achieved in some higher plants. Here, we provide an overview of SSN-facilitated GT in higher plants and discuss the potential of GT as a powerful tool for generating crop plants with desirable features.

Cell division and cell allocation in early mouse development

Development ◽  
1978 ◽  
Vol 48 (1) ◽  
pp. 37-51
Author(s):  
S. J. Kelly ◽  
J. G. Mulnard ◽  
C. F. Graham
Keyword(s):  
Cell Division ◽  
Tritiated Thymidine ◽  
Mouse Embryos ◽  
Mouse Development ◽  
Stages Of Development ◽  
Cell Stage ◽  
Cell Cycles ◽  
Short Cell ◽  
Early Mouse

Cell division was observed in intact and dissociated mouse embryos between the 2-cell stage and the blastocyst in embryos developing in culture. Division to the 4-cell stage was usually asynchronous. The first cell to divide to the 4-cell stage produced descendants which tended to divide ahead of those cells produced by its slow partner at all subsequent stages of development up to the blastocyte stage. The descendants of the first cell to divide to the 4-cell stage did not subsequently have short cell cycles. The first cell or last cell to divide from the 4-cell stage was labelled with tritiated thymidine. The embryo was reassembled, and it was found that the first pair of cells to reach the 8-cell stage contributed disproportionately more descendants to the ICM when compared with the last cell to divide to the 8-cell stage.

Distribution of microvilli on dissociated blastomeres from mouse embryos: evidence for surface polarization at compaction

Development ◽  
1981 ◽  
Vol 62 (1) ◽  
pp. 339-350
Author(s):  
W. J. D. Reeve ◽  
C. A. Ziomek
Keyword(s):  
Ligand Binding ◽  
Single Cells ◽  
Progressive Increase ◽  
Mouse Embryos ◽  
Cell Stage ◽  
Characteristic Distribution ◽  
Surface Polarization ◽  
Cell Embryo ◽  
Fluorescent Ligand ◽  
Scanning Electron

Cells of mouse embryos develop a polarization of microvillous distribution at compaction. Cells of the 4-cell embryo show a uniform pattern of fluorescent-ligand binding and an even distribution of microvilli. Each cell of the early 8-cell embryo has a uniform distribution both of microvilli and of fluorescent ligand. During the 8-cell stage, there is a progressive increase in the incidence of cells which show microvilli restricted to a region normally on the exposed surface of the embryo. When late 8-cell embryos were disaggregated to single cells, and these sorted by pattern of fluorescent-ligand binding, each of the four patterns of staining related consistently to a characteristic distribution of microvilli as viewed by scanning electron microscopy. The 16-cell embryo possessed an inside population of uniformly labelled cells with a sparse microvillous distribution, and an outside population of cells, each of which had a microvillous pole.

Quantitative aspects of RNA synthesis and polyadenylation in 1-cell and 2-cell mouse embryos

Development ◽  
1983 ◽  
Vol 74 (1) ◽  
pp. 169-182
Author(s):  
Kerry B. Clegg ◽  
Lajos Pikó
Keyword(s):  
Time Course ◽  
Specific Activity ◽  
Average Length ◽  
State Level ◽  
Mouse Embryos ◽  
High Rate ◽  
Rna Sequences ◽  
Cell Stage ◽  
New Synthesis ◽  
Cell Embryo

Mouse embryos at the late 1-cell and late 2-cell stages were labelled with [3H]adenosine for periods of up to 320 min during which the specific activity of the ATP pool was constant. The time course of the molar accumulation of adenosine was calculated for tRNA, high-molecular-weight poly(A)− RNA and poly(A) tails versus internal regions of poly(A)+ RNA. Most of the adenosine incorporation into tRNA is due to turnover of the 3′-terminal AMP but some new synthesis of tRNA also appears to take place in both 1-cell and 2-cell embryos at a rate of about 0·2 pg/embryo/h. In the poly(A)- RNA fraction, an unstable component which is assumed to be heterogeneous nuclear RNA is synthesized at a high rate and accumulates at a steady-state level of about 1·5 pg/embryo in the 1-cell embryo and about 3·0 pg/embryo in the 2-cell embryo. Both 1-cell and 2-cell embryos synthesize relatively stable heterogeneous poly(A)− RNA, assumed to be mRNA, at a rate of about 0·3 pg/embryo/h; 2-cell embryos also synthesize mature ribosomal RNA at a rate of about 0·4 pg/embryo·h. Internally labelled poly(A)+ RNA is synthesized at a low rate in the 1-cell embryo, about 0·045 pg/embryo/h, but the rate increases to about 0·2 pg/embryo/h by the 2-cell stage. A striking feature of the 1-cell embryo is the high rate of synthesis of poly(A) tails, about 2·5 × 106 tails/embryo/h of an average length of (A)43, due almost entirely to cytoplasmic polyadenylation. This and other evidence suggests a turnover of the poly(A)+ RNA population in 1-cell embryos as a result of polyadenylation of new RNA sequences and degradation of some of the pre-existing poly(A)+ RNA. In the 2-cell embryo, the rate of synthesis of poly(A) tails (average length (A)93) is estimated at about 0·8 × 106tails/embryo/h and a significant fraction of poly(A) synthesis appears to be nuclear.

Postimplantation development of tetraploid mouse embryos produced by electrofusion

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1121-1132
Author(s):  
M.H. Kaufman ◽  
S. Webb
Keyword(s):  
Cytochalasin B ◽  
Histological Analysis ◽  
Mouse Embryos ◽  
Normal Embryo ◽  
Craniofacial Abnormalities ◽  
Success Rates ◽  
Cell Stage ◽  
Extraembryonic Membranes ◽  
Mammalian Embryonic Development ◽  
Viable Embryo

Despite the fact that a variety of experimental techniques have been devised over the years to induce tetraploid mammalian embryonic development, success rates to date have been limited. Apart from the early study by Snow, who obtained development to term of a limited number of cytochalasin B-induced tetraploid mouse embryos, no other researchers have achieved development of tetraploid embryos beyond the early postimplantation period. We now report advanced postimplantation development of tetraploid mouse embryos following electrofusion of blastomeres at the 2-cell stage, and subsequent transfer of these 1-cell ‘fused’ embryos to appropriate recipients. Cytogenetic analysis of the extraembryonic membranes of all of the postimplantation embryos encountered in the present study has provided an unequivocal means of confirming their tetraploid chromosome constitution. A preliminary morphological and histological analysis of the tetraploid embryos obtained by this technique has revealed that characteristic craniofacial abnormalities particularly involving the forebrain and eyes were consistently observed, and these features were often associated with abnormalities of the vertebral axis and heart. The most advanced viable embryo in this series was recovered on the 15th day of gestation, and its morphological features suggest that it was developmentally equivalent to a normal embryo of about 13.5-14 days p.c.

Sign in / Sign up

Export Citation Format

Share Document