Efficient Generation of Mice with Consistent Transgene Expression by FEEST

Abstract Transgenic mouse models are widely used in biomedical research; however, current techniques for producing transgenic mice are limited due to the unpredictable nature of transgene expression. Here, we report a novel, highly efficient technique for the generation of transgenic mice with single-copy integration of the transgene and guaranteed expression of the gene-of-interest (GOI). We refer to this technique as functionally enriched ES cell transgenics, or FEEST. ES cells harboring an inducible Cre gene enabled the efficient selection of transgenic ES cell clones using hygromycin before Cre-mediated recombination. Expression of the GOI was confirmed by assaying for the GFP after Cre recombination. As a proof-of-principle, we produced a transgenic mouse line containing Cre-activatable tTA (cl-tTA6). This tTA mouse model was able to induce tumor formation when crossed with a transgenic mouse line containing a doxycycline-inducible oncogene. We also showed that the cl-tTA6 mouse is a valuable tool for faithfully recapitulating the clinical course of tumor development. We showed that FEEST can be easily adapted for other genes by preparing a transgenic mouse model of conditionally activatable EGFR L858R. Thus, FEEST is a technique with the potential to generate transgenic mouse models at a genome-wide scale.

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Overexpression of spermidine/spermine N1-acetyltransferase under the control of mouse metallothionein I promoter in transgenic mice: evidence for a striking post-transcriptional regulation of transgene expression by a polyamine analogue

Biochemical Journal ◽

10.1042/bj3380311 ◽

1999 ◽

Vol 338 (2) ◽

pp. 311-316 ◽

Cited By ~ 17

Author(s):

Suvikki SUPPOLA ◽

Marko PIETILÄ ◽

Jyrki J. PARKKINEN ◽

Veli-Pekka KORHONEN ◽

Leena ALHONEN ◽

...

Keyword(s):

Enzyme Activity ◽

Transgenic Mice ◽

Transgenic Mouse ◽

Cell Injury ◽

Mrna Level ◽

Transgenic Animals ◽

Ultrastructural Changes ◽

Mouse Line ◽

Transgenic Mouse Line ◽

Polyamine Analogue

We recently generated a transgenic mouse line overexpressing spermidine/spermine N1-acetyltransferase (SSAT) gene under its own promoter. The tissue polyamine pools of these animals were profoundly affected and the mice were hairless from early age. We have now generated another transgenic-mouse line overexpressing the SSAT gene under the control of a heavy-metal-inducible mouse metallothionein I (MT) promoter. Even in the absence of heavy metals, changes in the tissue polyamine pools indicated that a marked activation of polyamine catabolism had occurred in the transgenic animals. As with the SSAT transgenic mice generated previously, the mice of the new line (MT-SSAT) suffered permanent hair loss, but this occurred considerably later than in the previous SSAT transgenic animals. Liver was the most affected tissue in the MT–SSAT transgenic animals, revealed by putrescine overaccumulation, significant decrease in spermidine concentration and > 90% reduction in the spermine pool. Even though hepatic SSAT mRNA accumulated to massive levels in non-induced transgenic animals, SSAT activity was only moderately elevated. Administration of ZnSO4 further elevated the level of hepatic SSAT message and induced enzyme activity, but not more than 2- to 3-fold. Treatment of the transgenic animals with the polyamine analogue N1,N11-diethylnorspermine (DENSPM) resulted in an immense induction, more than 40000-fold, of enzyme activity in the liver of transgenic animals, and minor changes in the SSAT mRNA level. Liver spermidine and spermine pools were virtually depleted within 1–2 days in response to the treatment with the analogue. The treatment also resulted in a marked mortality (up to 60%) among the transgenic animals which showed ultrastructural changes in the liver, most notably mitochondrial swelling, one of the earliest signs of cell injury. These results indicated that, even without its own promoter, SSAT is powerfully induced by the polyamine analogue through a mechanism that appears to involve a direct translational and/or heterogenous nuclear RNA processing control. It is likewise significant that overexpression of SSAT renders the animals extremely sensitive to polyamine analogues.

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Transgenic mouse models of Alzheimer's disease: phenotype and mechanisms of pathogenesis

Biochemical Society Symposium ◽

10.1042/bss0670195 ◽

2001 ◽

Vol 67 ◽

pp. 195-202 ◽

Cited By ~ 18

Author(s):

Karen Duff

Keyword(s):

Alzheimer’S Disease ◽

Alzheimer's Disease ◽

Cognitive Impairment ◽

Transgenic Mice ◽

Amyloid Precursor Protein ◽

Transgenic Mouse ◽

Mouse Models ◽

Precursor Protein ◽

Transgenic Mouse Models ◽

Disease Phenotype

A range of transgenic mice have been created to model Alzheimer's disease. These include mice expressing human forms of the amyloid precursor protein, the presenilins and, more recently, tau. Several of the models develop features of the disease including amyloid pathology, cholinergic deficits, neurodegeneration and cognitive impairment. Progress in the characterization and use of these model animals is discussed.

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Isolation and Analysis of a Genome-Edited Single-Hepatocyte from a Cas9 Transgenic Mouse Line

Methods in Molecular Biology - Microinjection ◽

10.1007/978-1-4939-8831-0_15 ◽

2018 ◽

pp. 257-271

Author(s):

Takayuki Sakurai ◽

Akiko Kamiyoshi ◽

Masato Ohtsuka ◽

Channabasavaiah B. Gurumurthy ◽

Masahiro Sato ◽

...

Keyword(s):

Transgenic Mouse ◽

Mouse Line ◽

Transgenic Mouse Line ◽

A Genome

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Comparison of a new pmp22 transgenic mouse line with other mouse models and human patients with CMT1A*

Journal of Anatomy ◽

10.1046/j.1469-7580.2002.00039.x ◽

2002 ◽

Vol 200 (4) ◽

pp. 377-390 ◽

Cited By ~ 56

Author(s):

A. M. Robertson ◽

J. Perea ◽

A. McGuigan ◽

R. H. M. King ◽

J. R. Muddle ◽

...

Keyword(s):

Transgenic Mouse ◽

Mouse Models ◽

Mouse Line ◽

Transgenic Mouse Line

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Kill two birds with one stone: making multi-transgenic pre-diabetes mouse models through insulin resistance and pancreatic apoptosis pathogenesis

PeerJ ◽

10.7717/peerj.4542 ◽

2018 ◽

Vol 6 ◽

pp. e4542 ◽

Cited By ~ 2

Author(s):

Siyuan Kong ◽

Jinxue Ruan ◽

Kaiyi Zhang ◽

Bingjun Hu ◽

Yuzhu Cheng ◽

...

Keyword(s):

Insulin Resistance ◽

Type 2 Diabetes ◽

Insulin Secretion ◽

Transgenic Mice ◽

Transgenic Mouse ◽

Mouse Models ◽

Pancreatic Islet ◽

Early Stage ◽

Transgenic Mouse Models

Background Type 2 diabetes is characterized by insulin resistance accompanied by defective insulin secretion. Transgenic mouse models play an important role in medical research. However, single transgenic mouse models may not mimic the complex phenotypes of most cases of type 2 diabetes. Methods Focusing on genes related to pancreatic islet damage, peripheral insulin resistance and related environmental inducing factors, we generated single-transgenic (C/EBP homology protein, CHOP) mice (CHOP mice), dual-transgenic (human islet amyloid polypeptide, hIAPP; CHOP) mice (hIAPP-CHOP mice) and triple-transgenic (11β-hydroxysteroid dehydrogenase type 1, 11β-HSD1; hIAPP; CHOP) mice (11β-HSD1-hIAPP- CHOP mice). The latter two types of transgenic (Tg) animals were induced with high-fat high-sucrose diets (HFHSD). We analyzed the diabetes-related symptoms and histology features of the transgenic animals. Results Comparing symptoms on the spot-checked points, we determined that the triple-transgene mice were more suitable for systematic study. The results of intraperitoneal glucose tolerance tests (IPGTT) of triple-transgene animals began to change 60 days after induction (p < 0.001). After 190 days of induction, the body weights (p < 0.01) and plasma glucose of the animals in Tg were higher than those of the animals in Negative Control (Nc). After sacrificed, large amounts of lipid were found deposited in adipose (p < 0.01) and ectopically deposited in the non-adipose tissues (p < 0.05 or 0.01) of the animals in the Tg HFHSD group. The weights of kidneys and hearts of Tg animals were significantly increased (p < 0.01). Serum C peptide (C-P) was decreased due to Tg effects, and insulin levels were increased due to the effects of the HFHSD in the Tg HFHSD group, indicating that damaged insulin secretion and insulin resistance hyperinsulinemia existed simultaneously in these animals. The serum corticosterone of Tg was slightly higher than those of Nc due to the effects of the 11βHSD-1 transgene and obesity. In Tg HFHSD, hepatic adipose deposition was more severe and the pancreatic islet area was enlarged under compensation, accompanying apoptosis. In the transgenic control diet (Tg ControlD) group, hepatic adipose deposition was also severe, pancreatic islets were damaged, and their areas were decreased (p < 0.05), and apoptosis of pancreatic cells occurred. Taken together, these data show the transgenes led to early-stage pathological changes characteristic of type 2 diabetes in the triple-transgene HFHSD group. The disease of triple-transgenic mice was more severe than that of dual or single-transgenic mice. Conclusion The use of multi-transgenes involved in insulin resistance and pancreatic apoptosis is a better way to generate polygene-related early-stage diabetes models.

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Transgenic mice over-producing putrescine in their tissues do not convert the diamine into higher polyamines

Biochemical Journal ◽

10.1042/bj2910505 ◽

1993 ◽

Vol 291 (2) ◽

pp. 505-508 ◽

Cited By ~ 25

Author(s):

M Halmekytö ◽

L Alhonen ◽

L Alakuijala ◽

J Jänne

Keyword(s):

Transgenic Mice ◽

Transgenic Mouse ◽

Ornithine Decarboxylase ◽

Specific Inhibitor ◽

Mouse Line ◽

Transgenic Mouse Line ◽

Adenosylmethionine Decarboxylase ◽

Adult Tissues ◽

Ornithine Transaminase

We recently described a transgenic mouse line over-expressing the human ornithine decarboxylase gene virtually in all tissues. Despite strikingly elevated tissue putrescine concentrations, no or minimal changes were found in the levels of the higher polyamines spermidine and spermine. We have now extended these studies by further increasing tissue putrescine with the aid of 5-fluoromethylornithine, a specific inhibitor of ornithine transaminase and hence the catabolism of L-ornithine. As a result of the treatment with the latter drug, the concentration of putrescine was further increased by a factor of 2-3 without any changes in the concentrations of spermidine and spermine. In the testis of transgenic mice treated with 5-fluoromethylornithine, the concentration of putrescine was nearly 60 times that in non-transgenic untreated animals, yet the concentration of spermidine was only 1.5-fold higher. A similar small increase in brain spermidine was accompanied by a 40-fold elevation in the concentration of putrescine. The apparent blockade between putrescine and spermidine was in all likelihood not attributable to an inhibition of S-adenosylmethionine decarboxylase, the rate-controlling enzyme in the biosynthesis of spermidine and spermine. Our results are more compatible with the view that in non-dividing adult tissues putrescine is sequestered through some unknown mechanisms in a way that makes it unavailable for the synthesis of the higher polyamines.

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Overexpression of the Cdk5 inhibitory peptide in motor neurons rescue of amyotrophic lateral sclerosis phenotype in a mouse model

Human Molecular Genetics ◽

10.1093/hmg/ddz118 ◽

2019 ◽

Vol 28 (19) ◽

pp. 3175-3187

Author(s):

Binukumar BK ◽

Susan Skuntz ◽

Michaela Prochazkova ◽

Sashi Kesavapany ◽

Niranjana D Amin ◽

...

Keyword(s):

Spinal Cord ◽

Amyotrophic Lateral Sclerosis ◽

Mouse Model ◽

Transgenic Mouse ◽

Motor Neurons ◽

Motor Nerve ◽

Mouse Line ◽

Inhibitory Peptide ◽

Transgenic Mouse Line ◽

Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor nerve cells in the brain and the spinal cord. Etiological mechanisms underlying the disease remain poorly understood; recent studies suggest that deregulation of p25/Cyclin-dependent kinase 5 (Cdk5) activity leads to the hyperphosphorylation of Tau and neurofilament (NF) proteins in ALS transgenic mouse model (SOD1G37R). A Cdk5 involvement in motor neuron degeneration is supported by analysis of three SOD1G37R mouse lines exhibiting perikaryal inclusions of NF proteins and hyperphosphorylation of Tau. Here, we tested the hypothesis that inhibition of Cdk5/p25 hyperactivation in vivo is a neuroprotective factor during ALS pathogenesis by crossing the new transgenic mouse line that overexpresses Cdk5 inhibitory peptide (CIP) in motor neurons with the SOD1G37R, ALS mouse model (TriTg mouse line). The overexpression of CIP in the motor neurons significantly improves motor deficits, extends survival and delays pathology in brain and spinal cord of TriTg mice. In addition, overexpression of CIP in motor neurons significantly delays neuroinflammatory responses in TriTg mouse. Taken together, these data suggest that CIP may serve as a novel therapeutic agent for the treatment of neurodegenerative diseases.

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Toward a transgenic mouse model of remyelination

Multiple Sclerosis Journal ◽

10.1177/135245859700300204 ◽

1997 ◽

Vol 3 (2) ◽

pp. 80-83 ◽

Cited By ~ 3

Author(s):

S. Ferraresi ◽

I. Lorenzetti ◽

R. Nemni ◽

J. Kamholz ◽

ML Feltri ◽

...

Keyword(s):

Growth Factor ◽

Mouse Model ◽

Transgenic Mice ◽

Fibroblast Growth Factor ◽

Transgenic Mouse ◽

Molecular Mechanisms ◽

Basic Protein ◽

Transgenic Mouse Model ◽

Fibroblast Growth ◽

Transgenic Mouse Models

The molecular mechanisms necessary for remyelination by oligodendrocytes remain unexplored. We previously characterized a myelin basic protein promoter-lacZ (MBP-lacZ) transgene whose expression is regulated uniquely during development, and also in pathological situations, suggesting that it may be a useful reporter of molecular mechanisms during remyelination. As a first step toward creating a transgenic mouse model of remyelination, we cultured oligodendrocytes from these transgenic mice and showed that expression of MBP-lacZ appeared in parallel with a marker of oligodendrocyte maturation, galactocerebroside (GC). In addition, basic fibroblast growth factor blocked the expression of both MBP-lacZ and GC in these cells. Therefore, expression of MBP-lacZ reflects not only the developmental stage of oligodendrocytes, but also extrinsic influences on oligodendrocytes. These data suggest that MBP-lacZ may be a useful marker in transgenic mouse models of remyelination.

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Gα13 loss promotes tumor progression in the KPC transgenic mouse model of advanced pancreatic cancer

10.1101/2021.03.15.435488 ◽

2021 ◽

Author(s):

Mario A Shields ◽

Christina Spaulding ◽

Mahmoud G Khalafalla ◽

Thao ND Pham ◽

Hidayatullah G Munshi

Keyword(s):

Pancreatic Cancer ◽

Mouse Model ◽

Transgenic Mouse ◽

Mouse Models ◽

Cell Lines ◽

Advanced Pancreatic Cancer ◽

Transgenic Mouse Model ◽

Mtor Signaling ◽

Transgenic Mouse Models ◽

E Cadherin

Gα13 transduces signals from G protein-coupled receptors. Gα13 is pro-tumorigenic in epithelial cancer cell lines, which contrasts with its tumor-suppressive function in transgenic mouse models of lymphomas. Here we show that while loss of Gα13 in pancreatic cell lines decreases tumor growth in vivo, Gα13 loss in the Kras-driven (KC) mouse model of pancreatic tumor initiation does not affect tumor development or survival. Instead, Gα13 loss in the Kras/Tp53 (KPC) transgenic mouse model of advanced pancreatic cancer promotes well-differentiated tumors with increased tumor burden and reduced survival. Mechanistically, Gα13 loss in the KPC mouse model enhances E-cadherin-mediated cell-cell junctions and mTOR signaling. Importantly, human pancreatic cancers with low Gα13 expression exhibit increased E-cadherin protein expression and mTOR signaling. This work establishes a context-dependent role of Gα13 in pancreatic tumorigenesis, demonstrating a tumor-suppressive role in transgenic mouse models of advanced pancreatic cancer.

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