Generation of Large Fragment Knock-In Mouse Models by Microinjecting into 2-Cell Stage Embryos

2019 ◽  
pp. 89-100
Author(s):  
Bin Gu ◽  
Marina Gertsenstein ◽  
Eszter Posfai
Keyword(s):  
Mouse Models ◽  
Large Fragment ◽  
Cell Stage

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.

Cell-matrix interactions in the early mouse embryo

1992 ◽  
Vol 50 (1) ◽  
pp. 600-601
Author(s):  
A.E. Sutherland ◽  
P.G. Calarco ◽  
C.H. Damsky
Keyword(s):  
Mouse Embryo ◽  
Giant Cells ◽  
Blastocyst Stage ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Cell Stage ◽  
Early Mouse Embryo ◽  
Migratory Activity ◽  
Early Mouse ◽  
Cell Matrix Interactions

Cell-extracellular matrix (ECM) interactions mediated by the integrin family of receptors are critical for morphogenesis and may also play a regulatory role in differentiation during early development. We have examined the onset of expression of individual integrin subunit proteins in the early mouse embryo, and their roles in early morphogenetic events. As detected by immunoprecipitation, the α6, αV, β1, and β3 subunits are detected as early as the 4-cell stage, α5 at the hatched blastocyst stage and αl and α3 following blastocyst attachment. We tested the role of these integrins in the attachment and migratory activity of two cell populations of the early mouse embryo: the trophoblast giant cells, which invade the uterine stroma and ultimately contribute to the chorio-allantoic placenta, and the parietal endoderm, which migrates over the inner surface of the trophoblast and ultimately forms Reichert's membrane and the parietal yolk sac. Experiments were done in serum-free medium on substrates coated with laminin (Ln) and fibronectin (Fn). Trophoblast outgrowth occurs on Ln and its E8 fragment (long arm), but not on the E1’ fragment (cross region) (Figs. 1, 2 ). This outgrowth is inhibited by anti-E8, anti-Ln, and by the anti-β1 family antiserum anti-ECMR, but not by anti-αV or the function-perturbing GoH3 antibody that recognizes the α6/β1 integrin, a major Ln (E8) receptor. This suggests that trophoblast outgrowth on Ln or E8 is mediated by a different β1 integrin such as α3/β1. Early stages of trophoblast outgrowth (up to 48 hours) on Fn are inhibited by anti-Fn and by function-perturbing anti-αV antibodies, whereas at later times outgrowth becomes insensitive to anti-αV but remains sensitive to the anti-β1 family antiserum anti-ECMr, indicating that trophoblast cells modulate their interaction with Fn during outgrowth. Trophoblast outgrowth on vitronectin (Vn) is sensitive to anti-αV antibodies throughout the 5-day period examined.

Elemental Analysis of Phloem Tissue in Lemna Minor Root Tips

1980 ◽  
Vol 38 ◽  
pp. 798-799
Author(s):  
Patrick Echlin ◽  
Thomas Hayes ◽  
Clifford Lai ◽  
Greg Hook
Keyword(s):  
Vascular Tissue ◽  
Lemna Minor ◽  
Atomic Weight ◽  
Companion Cell ◽  
Root Tips ◽  
Phloem Tissue ◽  
Cell Stage ◽  
Phloem Parenchyma ◽  
Parenchyma Cells ◽  
Xylem Element

Studies (1—4) have shown that it is possible to distinguish different stages of phloem tissue differentiation in the developing roots of Lemna minor by examination in the transmission, scanning, and optical microscopes. A disorganized meristem, immediately behind the root-cap, gives rise to the vascular tissue, which consists of single central xylem element surrounded by a ring of phloem parenchyma cells. This ring of cells is first seen at the 4-5 cell stage, but increases to as many as 11 cells by repeated radial anticlinal divisions. At some point, usually at or shortly after the 8 cell stage, two phloem parenchyma cells located opposite each other on the ring of cells, undergo an unsynchronized, periclinal division to give rise to the sieve element and companion cell. Because of the limited number of cells involved, this developmental sequence offers a relatively simple system in which some of the factors underlying cell division and differentiation may be investigated, including the distribution of diffusible low atomic weight elements within individual cells of the phloem tissue.

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