Gene Targeting of the Receptor-Like Protein Tyrosine Phosphatase Lar by Homologous Recombination in Mouse Embryonic Stem Cells

Author(s):  
Roel Schaapveld ◽  
Jan Schepens ◽  
Frank Oerlemans ◽  
Michel Streuli ◽  
Bé Wieringa ◽  
...  
2016 ◽  
Vol 425 (1-2) ◽  
pp. 95-102
Author(s):  
Zahra Shokati Eshkiki ◽  
Mohammad Hossein Ghahremani ◽  
Parisa Shabani ◽  
Sattar Gorgani Firuzjaee ◽  
Asie Sadeghi ◽  
...  

1996 ◽  
Vol 91 (2) ◽  
pp. 304-307 ◽  
Author(s):  
Wouter G. van Inzen ◽  
Maikel P. Peppelenbosch ◽  
Maria W.M. van den Brand ◽  
Leon G.J. Tertoolen ◽  
Siegfried de Laat

Blood ◽  
2011 ◽  
Vol 117 (7) ◽  
pp. 2157-2165 ◽  
Author(s):  
Xinqiang Huang ◽  
Eric Gschweng ◽  
Ben Van Handel ◽  
Donghui Cheng ◽  
Hanna K. A. Mikkola ◽  
...  

Abstract MicroRNAs (miRs) play an important role in cell differentiation and maintenance of cell identity, but relatively little is known of their functional role in modulating human hematopoietic lineage differentiation. Human embryonic stem cells (hESCs) provide a model system to study early human hematopoiesis. We differentiated hESCs by embryoid body (EB) formation and compared the miR expression profile of undifferentiated hESCs to CD34+ EB cells. miRs-126/126* were the most enriched of the 7 miRs that were up-regulated in CD34+ cells, and their expression paralleled the kinetics of hematopoietic transcription factors RUNX1, SCL, and PU.1. To define the role of miRs-126/126* in hematopoiesis, we created hESCs overexpressing doxycycline-regulated miRs-126/126* and analyzed their hematopoietic differentiation. Induction of miRs-126/126* during both EB differentiation and colony formation reduced the number of erythroid colonies, suggesting an inhibitory role of miRs-126/126* in erythropoiesis. Protein tyrosine phosphatase, nonreceptor type 9 (PTPN9), a protein tyrosine phosphatase that is required for growth and expansion of erythroid cells, is one target of miR-126. PTPN9 restoration partially relieved the suppressed erythropoiesis caused by miRs-126/126*. Our results define an important function of miRs-126/126* in negative regulation of erythropoiesis, providing the first evidence for a role of miR in hematopoietic differentiation of hESCs.


1998 ◽  
Vol 18 (7) ◽  
pp. 4070-4078 ◽  
Author(s):  
Greg Donoho ◽  
Maria Jasin ◽  
Paul Berg

ABSTRACT To investigate the effects of in vivo genomic DNA double-strand breaks on the efficiency and mechanisms of gene targeting in mouse embryonic stem cells, we have used a series of insertion and replacement vectors carrying two, one, or no genomic sites for the rare-cutting endonuclease I-SceI. These vectors were introduced into the hypoxanthine phosphoribosyltransferase (hprt) gene to produce substrates for gene-targeting (plasmid-to-chromosome) or intrachromosomal (direct repeat) homologous recombination. Recombination at the hprt locus is markedly increased following transfection with an I-SceI expression plasmid and a homologous donor plasmid (if needed). The frequency of gene targeting in clones with an I-SceI site attains a value of 1%, 5,000-fold higher than that in clones with no I-SceI site. The use of silent restriction site polymorphisms indicates that the frequencies with which donor plasmid sequences replace the target chromosomal sequences decrease with distance from the genomic break site. The frequency of intrachromosomal recombination reaches a value of 3.1%, 120-fold higher than background spontaneous recombination. Because palindromic insertions were used as polymorphic markers, a significant number of recombinants exhibit distinct genotypic sectoring among daughter cells from a single clone, suggesting the existence of heteroduplex DNA in the original recombination product.


1994 ◽  
Vol 14 (4) ◽  
pp. 2404-2410 ◽  
Author(s):  
H Zhang ◽  
P Hasty ◽  
A Bradley

We analyzed the gene targeting frequencies and recombination products generated by a series of replacement deletion vectors which target the hprt (hypoxanthine phosphoribosyltransferase) locus in mouse embryonic stem cells. We found that the targeting frequency of a 19.2-kb deletion was comparable to that of a 3-kb deletion or a conventional replacement event in which a 1.7-kb fragment was inserted into the locus. We also observed different integration patterns for these deletion vectors. A result of this finding is that a wide range of genomic deletions in embryonic stem cells is feasible.


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