Transgenic mice

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 938-949 ◽  
Author(s):  
C. Babinet ◽  
D. Morello ◽  
J. P. Renard
Keyword(s):  
Homologous Recombination ◽  
Transgenic Mice ◽  
Dna Sequences ◽  
Insertional Mutagenesis ◽  
Germ Line ◽  
Embryonic Stem ◽  
Physiological Role ◽  
Cis Acting ◽  
Endogenous Genes

Stable integration into the mouse genome of exogenous genetic information has become, over the past few years, a very potent approach for different aspects of biology. It is a common feature that the integrated exogenous gene (the transgene) is expressed properly both spatially and temporally. Constructing different lines of transgenic mice carrying various versions of a gene, therefore, permits cis acting DNA sequences involved in the specificity of expression to be defined, in the context of the developing animal. This in turn opens the way to a variety of experiments in which a given gene product is targeted to one or another cell type, thus offering some insight into the physiological role of this product. Such a strategy has been used, for example, to address the questions of the role of oncogenes in malignant transformation. The insertion of foreign DNA per se may disrupt the function of endogenous genes, thus creating an insertional mutation. The corresponding affected genes may subsequently be cloned, using the transgene as a tag. Finally, the ability to perform homologous recombination, recently demonstrated with embryonic stem cells that can colonize the germ line of a foreign embryo, should constitute in the near future a unique way to analyse in detail the functioning of the mammalian genome.Key words: transgenic mice, oncogenes, insertional mutagenesis, cis-acting sequences, homologous recombination.

scholarly journals Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice

1993 ◽  
Vol 90 (23) ◽  
pp. 11381-11385 ◽  
Author(s):  
K M Gaensler ◽  
M Kitamura ◽  
Y W Kan
Keyword(s):  
Transgenic Mice ◽  
Yeast Artificial Chromosome ◽  
Germ Line ◽  
Physiological Role ◽  
Artificial Chromosome ◽  
Chromatin Domain ◽  
Globin Genes ◽  
Beta Globin ◽  
Cis Acting ◽  
Artificial Chromosomes

Sequential expression of the genes of the human beta-globin locus requires the formation of an erythroid-specific chromatin domain spanning > 200 kb. Regulation of this gene family involves both local interactions with proximal cis-acting sequences and long-range interactions with control elements upstream of the locus. To make it possible to analyze the interactions of cis-acting sequences of the human beta-globin locus in their normal spatial and sequence context, we characterized two yeast artificial chromosomes (YACs) 150 and 230 kb in size, containing the entire beta-globin locus. We have now successfully integrated the 150-kb YAC into the germ line of transgenic mice as a single unrearranged fragment that includes the locus control region, structural genes, and 30 kb of 3' flanking sequences present in the native locus. Expression of the transgenic human beta-globin locus is tissue- and developmental stage-specific and closely follows the pattern of expression of the endogenous mouse beta-globin locus. By using homology-directed recombination in yeast and methods for the purification and transfer of YACs into transgenic mice, it will now be feasible to study the physiological role of cis-acting sequences in specifying an erythroid-specific chromatin domain and directing expression of beta-globin genes during ontogeny.

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.

Regulated expression of a murine class I gene in transgenic mice

1986 ◽  
Vol 6 (4) ◽  
pp. 1339-1342
Author(s):  
C Bieberich ◽  
G Scangos ◽  
K Tanaka ◽  
G Jay
Keyword(s):  
Transgenic Mice ◽  
Germ Line ◽  
Inbred Mice ◽  
Class I ◽  
Endogenous Gene ◽  
Histocompatibility Complex ◽  
Unique System ◽  
Regulated Expression ◽  
I Gene

The major histocompatibility complex class I genes play an essential role in the immune presentation of aberrant cells. To gain further insight into the regulation of the expression of these class I genes and to better define the functions of their protein products, we made use of the technique of gene transfer into the germ line of inbred mice. With the use of locus-specific DNA probes, we observed that a transgenic class I gene was expressed in a tissue-dependent fashion analogous to that of an endogenous class I gene. In addition, the level of expression of the transgenic gene was substantially higher that that of the endogenous gene. The availability of transgenic mice properly expressing a foreign murine class I gene provides a unique system to further define the role of the class I antigens in the maturation of the immune response and in determining the malignant and metastatic phenotypes of tumor cells.

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.

scholarly journals Impaired mucin synthesis and bicarbonate secretion in the colon of NHE8 knockout mice

2012 ◽  
Vol 303 (3) ◽  
pp. G335-G343 ◽  
Author(s):  
Hua Xu ◽  
Bo Zhang ◽  
Jing Li ◽  
Chunhui Wang ◽  
Huacong Chen ◽  
...  
Keyword(s):  
Embryonic Stem ◽  
Physiological Role ◽  
Normal Serum ◽  
Sodium Absorption ◽  
Bicarbonate Secretion ◽  
Embryonic And Fetal Development ◽  
Sodium Hydrogen Exchanger ◽  
Important Player ◽  
Normal Serum Sodium

Sodium/hydrogen exchanger 8 (NHE8), the newest member of the SLC9 family, is expressed at the apical membrane of the epithelial cells in the intestine and the kidney. Although NHE8 has been shown to be an important player for intestinal sodium absorption early in development, its physiological role in the intestine remains unclear. Here, we successfully created a NHE8 knockout (NHE8−/−) mouse model to study the function of this transporter in the intestinal tract. Embryonic stem cells containing interrupted NHE8 gene were injected into mouse blastocyst to produce NHE8+/− chimeras. NHE8−/− mice showed no lethality during embryonic and fetal development. These mice had normal serum sodium levels and no signs of diarrhea. Apically expressed NHE2 and NHE3 were increased in the small intestine of the NHE8−/− mice in compensation. The number of goblet cells and mucin (MUC)-positive cells in the colon was reduced in NHE8−/− mice along with mucosal pH, MUC2 expression as well as downregulated in adenoma (DRA) expression. Therefore, the role of NHE8 in the intestine involves both sodium absorption and bicarbonate secretion.

scholarly journals Strictly co-isogenic C57BL/6J-Prnp−/− mice: A rigorous resource for prion science

2016 ◽  
Vol 213 (3) ◽  
pp. 313-327 ◽  
Author(s):  
Mario Nuvolone ◽  
Mario Hermann ◽  
Silvia Sorce ◽  
Giancarlo Russo ◽  
Cinzia Tiberi ◽  
...  
Keyword(s):  
Laboratory Mouse ◽  
Embryonic Stem ◽  
Physiological Role ◽  
Cellular Prion Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Phenotypic Characterization ◽  
Spongiform Encephalopathies ◽  
Demyelinating Peripheral Neuropathy

Although its involvement in prion replication and neurotoxicity during transmissible spongiform encephalopathies is undisputed, the physiological role of the cellular prion protein (PrPC) remains enigmatic. A plethora of functions have been ascribed to PrPC based on phenotypes of Prnp−/− mice. However, all currently available Prnp−/− lines were generated in embryonic stem cells from the 129 strain of the laboratory mouse and mostly crossed to non-129 strains. Therefore, Prnp-linked loci polymorphic between 129 and the backcrossing strain resulted in systematic genetic confounders and led to erroneous conclusions. We used TALEN-mediated genome editing in fertilized mouse oocytes to create the Zurich-3 (ZH3) Prnp-ablated allele on a pure C57BL/6J genetic background. Genomic, transcriptional, and phenotypic characterization of PrnpZH3/ZH3 mice failed to identify phenotypes previously described in non–co-isogenic Prnp−/− mice. However, aged PrnpZH3/ZH3 mice developed a chronic demyelinating peripheral neuropathy, confirming the crucial involvement of PrPC in peripheral myelin maintenance. This new line represents a rigorous genetic resource for studying the role of PrPC in physiology and disease.

scholarly journals Loss of the homologous recombination generad51leads to Fanconi anemia-like symptoms in zebrafish

2016 ◽  
Author(s):  
Jan Gregor Botthof ◽  
Ewa Bielczyk-Maczyńska ◽  
Lauren Ferreira ◽  
Ana Cvejic
Keyword(s):  
Bone Marrow ◽  
Homologous Recombination ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Tumor Development ◽  
Embryonic Stem ◽  
Dominant Negative ◽  
Hematopoietic Stem ◽  
Recombination Protein

AbstractRAD51is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant negative mutations. FA is a hereditary DNA repair disorder characterized by various congenital abnormalities, progressive bone marrow failure and cancer predisposition. In this paper, we describe the first viable vertebrate model ofRAD51loss. Zebrafishrad51loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to crosslinking agents and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Co-mutation ofp53was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment ofRAD51as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this novel zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.Significance statementThe homologous recombination protein RAD51 has been extensively studied in prokaryotes and lower eukaryotes. However, there is a significant lack of knowledge of the role of this protein and its regulation in anin-vivocontext in vertebrates. Here we report the first viable vertebrate mutant model ofrad51in zebrafish. These mutant fish enabled us to confirm for the first time the recently discovered role ofRAD51in Fanconi anemia pathogenesis. We report that p53 linked embryonic stem cell defects directly lead to hematological impairments later in life. Co-mutation ofrad51withp53rescues the observed hematological defects, but predisposes the fish to early tumor development. The application of this model opens new possibilities to advance Fanconi anemia drug discovery.

Retrovirus-induced insertional mutagenesis: mechanism of collagen mutation in Mov13 mice

1991 ◽  
Vol 11 (10) ◽  
pp. 5154-5163
Author(s):  
D D Barker ◽  
H Wu ◽  
S Hartung ◽  
M Breindl ◽  
R Jaenisch
Keyword(s):  
Dna Sequences ◽  
Insertional Mutagenesis ◽  
De Novo ◽  
Induced Mutations ◽  
Cis Acting ◽  
First Intron ◽  
Regulatory Dna Sequences ◽  
Regulatory Dna ◽  
Collagen Promoter ◽  
Murine Leukemia

The Mov13 mouse strain carries a mutation in the alpha 1(I) procollagen gene which is due to the insertion of a Moloney murine leukemia provirus into the first intron. This insertion results in the de novo methylation of the provirus and flanking DNA, the alteration of chromatin structure, and the transcriptional inactivity of the collagen promoter. To address the mechanism of mutagenesis, we reintroduced a cloned and therefore demethylated version of the Mov13 mutant allele into mouse fibroblasts. The transfected gene was not transcribed, indicating that the transcriptional defect was not due to the hypermethylation. Rather, this result strongly suggests that the mutation is due to the displacement or disruption of cis-acting regulatory DNA sequences within the first intron. We also constructed a Mov13 variant allele containing a single long terminal repeat instead of the whole provirus. This construct also failed to express mRNA, indicating that the Mov13 mutation does not revert by provirus excision as has been observed for other retrovirus-induced mutations.

Human erythropoietin gene expression in transgenic mice: multiple transcription initiation sites and cis-acting regulatory elements

1990 ◽  
Vol 10 (3) ◽  
pp. 930-938
Author(s):  
G L Semenza ◽  
R C Dureza ◽  
M D Traystman ◽  
J D Gearhart ◽  
S E Antonarakis
Keyword(s):  
Transgenic Mice ◽  
Dna Sequences ◽  
Transcription Initiation ◽  
Cobalt Chloride ◽  
Fetal Liver ◽  
Regulatory Element ◽  
Regulatory Elements ◽  
Flanking Sequence ◽  
Rnase Protection ◽  
Cis Acting

Erythropoietin (EPO) is the primary humoral regulator of mammalian erythropoiesis. The single-copy EPO gene is normally expressed in liver and kidney, and increased transcription is induced by anemia or cobalt chloride administration. To identify cis-acting DNA sequences responsible for regulated expression, transgenic mice were generated by microinjection of a 4-kilobase-pair (kb) (tgEPO4) or 10-kb (tgEPO10) cloned DNA fragment containing the human EPO gene, 0.7 kb of 3'-flanking sequence, and either 0.4 or 6 kb of 5'-flanking sequence, respectively. tgEPO4 mice expressed the transgene in liver, where expression was inducible by anemia or cobalt chloride, kidney, where expression was not inducible, and other tissues that do not normally express EPO. Human EPO RNA in tgEPO10 mice was detected only in liver of anemic or cobalt-treated mice. Both tgEPO4 and tgEPO10 mice were polycythemic, demonstrating that the human EPO RNA transcribed in liver is functional. These results suggest that (i) a liver inducibility element maps within 4 kb encompassing the gene, 0.4 kb of 5'-flanking sequence, and 0.7 kb of 3'-flanking sequence; (ii) a negative regulatory element is located between 0.4 and 6 kb 5' to the gene; and (iii) sequences required for inducible kidney expression are located greater than 6 kb 5' or 0.7 kb 3' to the gene. RNase protection analysis revealed that human EPO RNA in anemic transgenic mouse liver and hypoxic human hepatoma cells is initiated from several sites, only a subset of which is utilized in nonanemic transgenic liver and human fetal liver.

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