Germ line transmission of yeast artificial chromosomes in transgenic mice

1994 ◽  
Vol 6 (5) ◽  
pp. 577 ◽  
Author(s):  
L Montoliu ◽  
A Schedl ◽  
G Kelsey ◽  
H Zentgraf ◽  
P Lichter ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Artificial Chromosomes ◽  
Tyrosinase Gene ◽  
Temporal And Spatial ◽  
Genomic Regions ◽  
Germ Line Transmission ◽  
Yeast Artificial Chromosomes

Several groups have recently reported the successful generation of transgenic mice harbouring yeast artificial chromosomes (YACs). Different methodological approaches have been shown to produce similar results, namely, the faithful expression of the transgenes carried on YAC DNA. In this paper, we compare the reported techniques for obtaining transgenic mice carrying YACs using a 250-kb YAC bearing the mouse tyrosinase gene. These methods include: microinjection of gel-purified YAC DNA into pronuclei of fertilized mouse oocytes, yeast spheroblast fusion with embryonic stem (ES) cells and lipofection of YAC DNA into ES cells. Taken together, these reports show that the delivery of large genomic regions covering a gene of interest (such as those cloned in YAC vectors) is feasible, and will ensure appropriate temporal and spatial expression of the transgene at a level comparable to that of the endogenous counterpart.

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity

Development ◽  
1990 ◽  
Vol 110 (4) ◽  
pp. 1341-1348 ◽  
Author(s):  
J. Nichols ◽  
E.P. Evans ◽  
A.G. Smith
Keyword(s):  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Leukaemia Inhibitory Factor ◽  
Inhibiting Activity ◽  
Es Cell ◽  
Essential Function ◽  
Germ Line Transmission

The regulatory factor Differentiation Inhibiting Activity/Leukaemia Inhibitory Factor (DIA/LIF) suppresses the differentiation of cultured embryonic stem (ES) cells. In the present study, it is shown that ES cell lines can be derived and maintained in the absence of feeder layers using medium supplemented with purified DIA/LIF. These cells can differentiate normally in vitro and in vivo and they retain the capacity for germ-line transmission. DIA/LIF therefore fulfils the essential function of feeders in the isolation of pluripotential stem cells.

Germ-line transmission of murine embryonic stem (ES) cells after targeted inactivation of one allele of the CNTF gene

1993 ◽  
Vol 39 (1) ◽  
pp. 341 ◽  
Author(s):  
E. Wolf ◽  
Y. Masu ◽  
H. Thoenen ◽  
G. Brem
Keyword(s):  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Germ Line Transmission ◽  
Targeted Inactivation

scholarly journals Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells.

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758 ◽  
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

scholarly journals Yeast artificial chromosomes: an alternative approach to the molecular analysis of mouse developmental mutations

1990 ◽  
Vol 56 (2-3) ◽  
pp. 203-208 ◽  
Author(s):  
Zoia Larin ◽  
Hans Lehrach
Keyword(s):  
Mouse Genome ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Artificial Chromosome ◽  
Molecular Study ◽  
Insert Size ◽  
Artificial Chromosomes ◽  
Partial Digestion ◽  
Genomic Regions ◽  
Yeast Artificial Chromosomes

SummaryMammalian genetics now allows a molecular study of genomic regions previously analysed by genetic and embryological techniques. To simplify such an analysis, we have established a number of libraries of mouse DNA in Yeast Artificial Chromosome (YAC) vectors, constructed either by partial digestion with EcoRI, or by complete digestion with enzymes which cut rarely in the mammalian genome. In this paper we report the construction of complete digest libraries prepared from mouse genomic DNA using the rare cutter enzymes NoiI and BssHII, and the detection of gene loci from the H-2 complex, the t–complex, and other loci from the mouse genome. Due to their large insert size, YAC clones simplify the cloning of extended regions of the mouse genome surrounding known developmental mutations and should, after introduction into the germ line, offer a high probability of correct expression of the genes contained within the cloned region. We hope that this will allow the use of YAC clones to scan regions of interest such as the t–complex for specific genes by testing DNA introduced into transgenic mice for the ability to complement mutations localised to this region.

scholarly journals Improved Experimental Procedures for Achieving Efficient Germ Line Transmission of Nonobese Diabetic (NOD)-Derived Embryonic Stem Cells

2004 ◽  
Vol 5 (3) ◽  
pp. 219-226 ◽  
Author(s):  
Satoko Arai ◽  
Christina Minjares ◽  
Seiho Nagafuchi ◽  
Toru Miyazaki
Keyword(s):  
High Efficiency ◽  
Germ Line ◽  
Gene Knockout ◽  
Es Cells ◽  
Embryonic Stem ◽  
Nod Mice ◽  
Insulin Dependent Diabetes Mellitus ◽  
Transmission Efficiency ◽  
Dependent Diabetes Mellitus ◽  
Specific Gene

The manipulation of a specific gene in NOD mice, the best animal model for insulin-dependent diabetes mellitus (IDDM), must allow for the precise characterization of the functional involvement of its encoded molecule in the pathogenesis of the disease. Although this has been attempted by the cross-breeding of NOD mice with many gene knockout mice originally created on the 129 or C57BL/6 strain background, the interpretation of the resulting phenotype(s) has often been confusing due to the possibility of a known or unknown disease susceptibility locus (e.g.,Iddlocus) cosegregating with the targeted gene from the diabetes-resistant strain. Therefore, it is important to generate mutant mice on a pure NOD background by using NOD-derived embryonic stem (ES) cells. By using the NOD ES cell line established by Nagafuchi and colleagues in 1999 (FEBSLett., 455, 101–104), the authors reexamined various conditions in the context of cell culture, DNA transfection, and blastocyst injection, and achieved a markedly improved transmission efficiency of these NOD ES cells into the mouse germ line. These modifications will enable gene targeting on a “pure” NOD background with high efficiency, and contribute to clarifying the physiological roles of a variety of genes in the disease course of IDDM.

scholarly journals Target frequency and integration pattern for insertion and replacement vectors in embryonic stem cells

1991 ◽  
Vol 11 (9) ◽  
pp. 4509-4517
Author(s):  
P Hasty ◽  
J Rivera-Pérez ◽  
C Chang ◽  
A Bradley
Keyword(s):  
Homologous Recombination ◽  
Gene Targeting ◽  
Mammalian Cells ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Homologous Sequence ◽  
Reciprocal Recombination ◽  
Replacement Vector ◽  
Insertion Vector

Gene targeting has been used to direct mutations into specific chromosomal loci in murine embryonic stem (ES) cells. The altered locus can be studied in vivo with chimeras and, if the mutated cells contribute to the germ line, in their offspring. Although homologous recombination is the basis for the widely used gene targeting techniques, to date, the mechanism of homologous recombination between a vector and the chromosomal target in mammalian cells is essentially unknown. Here we look at the nature of gene targeting in ES cells by comparing an insertion vector with replacement vectors that target hprt. We found that the insertion vector targeted up to ninefold more frequently than a replacement vector with the same length of homologous sequence. We also observed that the majority of clones targeted with replacement vectors did not recombine as predicted. Analysis of the recombinant structures showed that the external heterologous sequences were often incorporated into the target locus. This observation can be explained by either single reciprocal recombination (vector insertion) of a recircularized vector or double reciprocal recombination/gene conversion (gene replacement) of a vector concatemer. Thus, single reciprocal recombination of an insertion vector occurs 92-fold more frequently than double reciprocal recombination of a replacement vector with crossover junctions on both the long and short arms.

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