scholarly journals Simple and straightforward construction of a mouse gene targeting vector using in vitro transposition reactions

2005 ◽  
Vol 33 (5) ◽  
pp. e52-e52 ◽  
Author(s):  
M. Aoyama
Keyword(s):  
Gene Targeting ◽  
Mouse Gene ◽  
Targeting Vector ◽  
In Vitro Transposition

Production of chimeras by blastocyst and morula injection of targeted ES cells

1999 ◽  
Author(s):  
Virginia Papaioannou ◽  
Randall Johnson
Keyword(s):  
Stem Cells ◽  
Gene Targeting ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryonic Cells ◽  
Cell Potential ◽  
Teratocarcinoma Cell ◽  
Normal Manner ◽  
Mammalian Embryos

The ability of mammalian embryos to incorporate foreign cells and develop as chimeras has been exploited for a variety of purposes including the elucidation of cell lineages, the investigation of cell potential, the perpetuation of mutations produced in embryonic stem (ES) cells by gene targeting, and the subsequent analysis of these mutations. The extent of contribution of the foreign cells depends on their developmental synchrony with the host embryo and their mitotic and developmental potential, which may be severely restricted if the cells bear mutations. If the goal in making chimeras is the transmission of a mutation produced by gene targeting to the next generation, the mutant ES cells must have the capacity to undergo meiosis and gametogenesis. Cells from two different mammalian embryos were first combined experimentally to produce a composite animal, dubbed a chimera, nearly four decades ago. Pairs of cleaving, pre-implantation embryos were mechanically associated in vitro until they aggregated together to make single large morulae; these in turn resulted in chimeric offspring (1). Genetic markers were used to distinguish the contributions of the two embryos in these animals. Since then, various methods for making chimeras have been explored to address different types of questions (2). In 1972 it was reported that highly asynchronous embryonic cells, which had been cultured in vitro, could contribute to chimeras upon re-introduction into pre-implantation embryos (3). Not long afterward, several groups working with teratocarcinomas, tumours derived from germ cells of the gonad, discovered that stem cells from these tumours, known as embryonal carcinoma cells, could contribute to an embryo if introduced into pre-implantation stages (4-6). It appeared that the undifferentiated stem cells of the tumour had enough features in common with early embryonic cells that they could respond to the embryonic environment, differentiating in a normal manner, even after long periods in vitro. Their embryonic potential was limited, however, and many teratocarcinoma cell lines made only meagre contributions to the developing fetus or even produced tumours in chimeras (7). Either their derivation from tumours or their extended sojourn in vitro rendered these cells so dissimilar from early embryonic cells that they rarely, if ever, had full embryonic potential.

scholarly journals Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition

2010 ◽  
Vol 11 (12) ◽  
pp. R119 ◽  
Author(s):  
Andrew Adey ◽  
Hilary G Morrison ◽  
Asan (no last name) ◽  
Xu Xun ◽  
Jacob O Kitzman ◽  
...  
Keyword(s):  
High Density ◽  
Low Input ◽  
Fragment Libraries ◽  
In Vitro Transposition

scholarly journals Ectopic gene targeting exhibits a bimodal distribution of integration in murine cells, indicating that both intra- and interchromosomal sites are accessible to the targeting vector.

1997 ◽  
Vol 17 (9) ◽  
pp. 5571-5580 ◽  
Author(s):  
G Dellaire ◽  
N Lemieux ◽  
A Belmaaza ◽  
P Chartrand
Keyword(s):  
Gene Targeting ◽  
Genetic Locus ◽  
Bimodal Distribution ◽  
New Paradigm ◽  
Target Locus ◽  
Fibroblast Line ◽  
Alternative Outcome ◽  
Murine Fibroblast ◽  
Integration Sites ◽  
Targeting Vector

Ectopic gene targeting is an alternative outcome of the gene targeting process in which the targeting vector acquires sequences from the genomic target but proceeds to integrate elsewhere in the genome. Using two-color fluorescent in situ hybridization analysis, we have determined the integration sites of the gene targeting vector with respect to the target locus in a murine fibroblast line (LTA). We found that for ectopic gene targeting the distribution of integration sites was bimodal, being either within 3 Mb of the target or on chromosomes distinct from the chromosome carrying the target locus. Inter- and intrachromosomal sites appeared to be equally accessible to the targeting vector, with site-specific variations. Interestingly, interphase analysis indicated that vector sequences which had integrated ectopically in chromosomes other than the target colocalized with the target locus at a significant frequency compared to that of colocalization to random unlinked loci. We propose that ectopic gene targeting could be used to determine which chromosomal domains within the genome are accessible to a given genetic locus. Thus, recombination access mapping may present a new paradigm for the analysis of DNA accessibility and interaction within the genome.

scholarly journals The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.

1990 ◽  
Vol 10 (7) ◽  
pp. 3456-3467 ◽  
Author(s):  
P Lemaire ◽  
C Vesque ◽  
J Schmitt ◽  
H Stunnenberg ◽  
R Frank ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Transcriptional Activator ◽  
Mouse Gene ◽  
Target Sequence ◽  
Recognition Sequence ◽  
Reading Frame ◽  
Cell Cycle Reentry ◽  
Vaccinia Viruses ◽  
Simplex Virus

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.

A novel promoterless gene targeting vector to efficiently disrupt PRNP gene in cattle

2013 ◽  
Vol 163 (4) ◽  
pp. 377-385 ◽  
Author(s):  
Shaohua Wang ◽  
Kun Zhang ◽  
Fangrong Ding ◽  
Rui Zhao ◽  
Song Li ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Prnp Gene ◽  
Targeting Vector
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