A 1,064 bp fragment from the promoter region of the Col11a2 gene drives lacz expression not only in cartilage but also in osteoblasts adjacent to regions undergoing both endochondral and intramembranous ossification in mouse embryos

Type XI collagen is a structural component of the cartilage extracellular matrix and plays an important role in skeletal morphogenesis. As a step toward defining the molecular mechanisms responsible for the regulation of type XI collagen expression, we characterized the promoter region of the mouse alpha 2(XI) collagen gene (Coll1a2). We also generated transgenic mice harboring various fragments of the promoter and the first intron of Coll1a2 linked to the Escherichia coli beta-galactosidase gene to identify the cis-acting elements responsible for tissue- and site-specific expression during development. Cloning and sequence analysis of the 5' flanking region of Coll1a2 showed that the putative 3' end of the retinoid X receptor beta gene was located 742 bp upstream of the Coll1a2 start site. This suggested that the promoter region of Coll1a2 was localized within this 742-bp sequence, which contained multiple consensus regulatory elements. Examination of the transgenic mice revealed that the longest DNA construct (containing the entire promoter and first intron sequences) directed lacZ expression in the notochord as well as in the primordial cartilage throughout the body, with the pattern of expression mimicking that of endogenous Coll1a2 transcripts. On the other hand, deletion of the upstream approximately 290 bp resulted in the elimination of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, whereas lacZ expression in the notochord and in the other primordial cartilage elsewhere was not affected. Deletion of the first intron sequence also resulted in the loss of lacZ expression in the primordial cartilage of the carpals, tarsals, and vertebral bodies, as well as in the notochord. These results demonstrate that the upstream 742-bp and first intron segments of the mouse Coll1a2 gene contain the necessary information to confer high level tissue-specific expression in mouse embryos. In addition, our observations suggest the presence of site-specific cis-acting elements that control Coll11a2 gene expression in different cartilaginous components of the skeleton.

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Additional enhancer copies, with intact cdx binding sites, anteriorize Hoxa-7/lacZ expression in mouse embryos: evidence in keeping with an instructional cdx gradient

The International Journal of Developmental Biology ◽

10.1387/ijdb.041829sg ◽

2004 ◽

Vol 48 (7) ◽

pp. 613-622 ◽

Cited By ~ 18

Author(s):

Stephen J. Gaunt ◽

Adam Cockley ◽

Deborah Drage

Keyword(s):

Binding Sites ◽

Mouse Embryos ◽

Lacz Expression

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Efficient Zygotic Genome Editing via RAD51-Enhanced Interhomolog Repair

10.1101/263699 ◽

2018 ◽

Cited By ~ 8

Author(s):

Jonathan J. Wilde ◽

Tomomi Aida ◽

Martin Wienisch ◽

Qiangge Zhang ◽

Peimin Qi ◽

...

Keyword(s):

Genome Editing ◽

Conclusive Evidence ◽

Mouse Embryos ◽

Strand Exchange ◽

Low Frequencies ◽

Gene Drives ◽

Potential Use ◽

Human Primate ◽

Stimulation Of ◽

Zygotic Genome

AbstractRecent advances in genome editing have greatly improved knock-in (KI) efficiency1–9. Searching for factors to further improve KI efficiency for therapeutic use and generation of non-human primate (NHP) models, we found that the strand exchange protein RAD51 can significantly increase homozygous KI using CRISPR/Cas9 in mouse embryos through an interhomolog repair (IHR) mechanism. IHR is well-described in the context of meiosis10, but only occurs at low frequencies in mitotic cells11,12 and its existence in zygotes is controversial. Using a variety of approaches, we provide evidence for an endogenous IHR mechanism in zygotes that can be enhanced by RAD51. We show that this process can be harnessed for generating homozygous KI animals from wildtype zygotes based on exogenous donors and for converting heterozygous alleles into homozygous alleles without exogenous templates. Furthermore, we elucidate additional factors that contribute to zygotic IHR and identify a RAD51 mutant capable of insertion-deletion (indel)-free stimulation of IHR. Thus, our study provides conclusive evidence for the existence of zygotic IHR and demonstrates methods to enhance IHR for potential use in gene drives, gene therapy, and biotechnology.

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Human Adenovirus Uptake by Preirnplantation Mouse Embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100094607 ◽

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.

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Preplate neurons in embryonic mouse cortex: Ultrastructural observations using photoconversion of the fluorescent lipophilic dye dil

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124938 ◽

1992 ◽

Vol 50 (1) ◽

pp. 906-907

Author(s):

Linda C. Hassinger ◽

James E. Crandall

Keyword(s):

Mouse Embryos ◽

Embryonic Mouse ◽

Cortical Afferents ◽

Mouse Cortex ◽

Carbocyanine Dye ◽

Increased Sensitivity

We have begun to look directly at small numbers of afferent axons to early generated neurons that form the preplate in the developing mouse cortex. The carbocyanine dye Dil (1’1, dioctadecyl-3,3,3’3’-tetramethyl-indocarbocyanine) has proved especially useful for this goal. DiI labels axons and their terminals with greater sensitivity and without some of the disadvantages of axon filling with HRP. The increased sensitivity provided by labeling embryonic axons with DiI has given us new insights into the development of cortical afferents. For instance, we reported originally that afferents from the thalamus were present below the cortex as early as embryonic day 15 (E15) based on HRP injections into mouse embryos. By using DiI placements into the thalamus in aldehyde-fixed brains, we now know that thalamic fibers reach the cortex 24 hrs earlier.

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Localization of Intracisternal a Particle Antigens in Neoplastic Cells and Preimplantation Mouse Embryos

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s1431927600003044 ◽

1980 ◽

Vol 38 ◽

pp. 696-697

Author(s):

Thomas T.F. Huang ◽

Patricia G. Calarco

Keyword(s):

Endoplasmic Reticulum ◽

Normal Development ◽

Mouse Embryos ◽

Neoplastic Cells ◽

Large Numbers ◽

Preimplantation Mouse Embryos ◽

Preimplantation Mouse ◽

Intracisternal A Particle ◽

Normal Feature

The stage specific appearance of a retravirus, termed the Intracisternal A particle (IAP) is a normal feature of early preimplantation development. To date, all feral and laboratory strains of Mus musculus and even Asian species such as Mus cervicolor and Mus pahari express the particles during the 2-8 cell stages. IAP form by budding into the endoplasmic reticulum and appear singly or as groups of donut-shaped particles within the cisternae (fig. 1). IAP are also produced in large numbers in several neoplastic cells such as certain plasmacytomas and rhabdomyosarcomas. The role of IAP, either in normal development or in neoplastic behavior, is unknown.

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