Robust Generation of Transgenic Mice by Hypotonic Shock Mediated Transgene Delivery in Testicular Germ Cells

2016 ◽  
Author(s):  
Abul Usmani ◽  
Nirmalya Ganguli ◽  
Subodh K Jain ◽  
Nilanjana Ganguli ◽  
Rajesh Kumar Sarkar ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Germ Cells ◽  
Transgenic Animals ◽  
Biological Research ◽  
Solution Ph ◽  
Functional Interpretation ◽  
Hypotonic Shock ◽  
New Genes ◽  
Transgene Delivery ◽  
Transgenic Progeny

AbstractOur ability to decipher gene sequences has increased enormously with the advent of modern sequencing tools but the ability to divulge functions of new genes have not increased correspondingly. This has caused a remarkable delay in functional interpretation of several newly found genes in tissue and age specific manner, limiting the pace of biological research. This is mainly due to lack of advancements in methodological tools for transgenesis. Predominantly practiced method of transgenesis by pronuclear DNA-microinjection is time consuming, tedious and requires highly skilled persons for embryo-manipulation. Testicular electroporation mediated transgenesis requires use of electric current to testis. To this end, we have now developed an innovative technique for making transgenic mice by giving hypotonic shock to male germ cells for the gene delivery. Desired transgene was suspended in hypotonic Tris-HCl solution (pH 7.0) and simply injected in testis. This resulted in internalization of the transgene in dividing germ-cells residing at basal compartment of tubules leading to its integration in native genome of mice. Such males generated transgenic progeny by natural mating. Several transgenic animals can be generated with minimum skill within short span of time by this easily adaptable novel technique.

scholarly journals Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

2008 ◽  
Vol 416 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Rafael Mayoral ◽  
Belen Mollá ◽  
Juana Maria Flores ◽  
Lisardo Boscá ◽  
Marta Casado ◽  
...  
Keyword(s):  
Liver Injury ◽  
Transgenic Mice ◽  
Genetic Model ◽  
Acute Liver Injury ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Hepatocyte Proliferation ◽  
Nuclear Antigen ◽  
Cox 2 ◽  
Inflammatory Profile

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.

scholarly journals The c-myc proto-oncogene regulates cardiac development in transgenic mice.

1990 ◽  
Vol 10 (7) ◽  
pp. 3709-3716 ◽  
Author(s):  
T Jackson ◽  
M F Allard ◽  
C M Sreenan ◽  
L K Doss ◽  
S P Bishop ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Mrna Expression ◽  
Cardiac Myocyte ◽  
Cellular Proliferation ◽  
Cardiac Development ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Hypertrophic Growth ◽  
Myocyte Proliferation

During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.

Meiotic expression of human ornithine transcarbamylase in the testes of transgenic mice

1988 ◽  
Vol 8 (4) ◽  
pp. 1821-1825
Author(s):  
K A Kelley ◽  
J W Chamberlain ◽  
J A Nolan ◽  
A L Horwich ◽  
F Kalousek ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fusion Gene ◽  
Regulatory Region ◽  
Transgenic Animals ◽  
Ornithine Transcarbamylase ◽  
Regulatory Sequences ◽  
Base Pairs ◽  
Protein Coding ◽  
Wide Range ◽  
Flanking Sequences

In an attempt to use mouse metallothionein-I (mMT-I) regulatory sequences to direct expression of human ornithine transcarbamylase in the liver of transgenic animals, fusion genes joining either 1.6 kilobases or 185 base pairs of the mMT-I regulatory region to the human ornithine transcarbamylase protein-coding sequence were used to produce transgenic mice. In mice carrying the fusion gene with 1.6 kilobases of the mMT-I 5'-flanking sequences, transgene expression was observed in a wide range of tissues, but, unexpectedly, expression in liver was never observed. Surprisingly, in mice carrying the fusion gene regulated by only 185 base pairs of the mMT-I 5'-flanking sequences, the transgene was expressed exclusively in male germ cells during the tetraploid, pachytene stage of meiosis.

Highly efficient lentiviral-mediated human cytokine transgenesis on the NOD/scid background

Blood ◽  
2004 ◽  
Vol 103 (2) ◽  
pp. 580-582 ◽  
Author(s):  
Isabel Punzon ◽  
Luis M. Criado ◽  
Alfredo Serrano ◽  
Fernando Serrano ◽  
Antonio Bernad
Keyword(s):  
Transgenic Mice ◽  
Lentiviral Vector ◽  
Cell Lineage ◽  
Transgenic Animals ◽  
Hematopoietic Tissue ◽  
Nonobese Diabetic ◽  
Human Blood Cell ◽  
Macrophage Colony ◽  
Human Cytokine

Abstract Human neo-organ formation from stem cells can only be assayed by in vivo xenotransplantation. The human nonobese diabetic–severe combined immunodeficient (HuNOD/scid) CD34+ cell transplantation is a model that allows examination of hematopoietic tissue formation, although human hematopoietic cell maturation is abortive. Conventional humanization of the cytokine microenvironment has depended on generation of human cytokine-transgenic mice in strains appropriate for conventional plasmid microinjection, followed by backcrossing, a costly and time-consuming approach. Lentiviral vector infection of single-cell embryos was recently reported to produce transgenic animals. Using this approach, we have generated direct human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic mice from lentivirus-microinjected NOD/scid embryos, with 68% efficiency and 100% penetrance; this allowed us to obtain NOD/scid transgenic mice with considerable savings of resources. This powerful technique should assist in producing novel mouse models for the study of human blood cell lineage development and other human neo-organs from stem cell xenotransplantation for which a similar “humanization” rationale may be required.

The c-myc proto-oncogene regulates cardiac development in transgenic mice

1990 ◽  
Vol 10 (7) ◽  
pp. 3709-3716
Author(s):  
T Jackson ◽  
M F Allard ◽  
C M Sreenan ◽  
L K Doss ◽  
S P Bishop ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Mrna Expression ◽  
Cardiac Myocyte ◽  
Cellular Proliferation ◽  
Cardiac Development ◽  
Constitutive Expression ◽  
Transgenic Animals ◽  
Hypertrophic Growth ◽  
Myocyte Proliferation

During the maturation of the cardiac myocyte, a transition occurs from hyperplastic to hypertrophic growth. The factors that control this transition in the developing heart are unknown. Proto-oncogenes such as c-myc have been implicated in the regulation of cellular proliferation and differentiation, and in the heart the switch from myocyte proliferation to terminal differentiation is synchronous with a decrease in c-myc mRNA abundance. To determine whether c-myc can influence myocyte proliferation or differentiation, we examined the in vivo effect of increasing c-myc expression during embryogenesis and of preventing the decrease in c-myc mRNA expression that normally occurs during cardiac development. The model system used was a strain of transgenic mice exhibiting constitutive expression of c-myc mRNA in cardiac myocytes throughout development. In these transgenic mice, increased c-myc mRNA expression was found to be associated with both atrial and ventricular enlargement. This increase in cardiac mass was secondary to myocyte hyperplasia, with the transgenic hearts containing more than twice as many myocytes as did nontransgenic hearts. The results suggest that in the transgenic animals there is additional hyperplastic growth during fetal development. However, this additional proliferative growth is not reflected in abnormal myocyte maturation, as assessed by the expression of the cardiac and skeletal isoforms of alpha-actin. The results of this study indicate that constitutive expression of c-myc mRNA in the heart during development results in enhanced hyperplastic growth and suggest a regulatory role for this proto-oncogene in cardiac myogenesis.

Inhibition of testicular germ cell apoptosis and differentiation in mice misexpressing Bcl-2 in spermatogonia

Development ◽  
1996 ◽  
Vol 122 (6) ◽  
pp. 1703-1709 ◽  
Author(s):  
T. Furuchi ◽  
K. Masuko ◽  
Y. Nishimune ◽  
M. Obinata ◽  
Y. Matsui
Keyword(s):  
Transgenic Mice ◽  
Germ Cells ◽  
Molecular Mechanisms ◽  
Elongation Factor ◽  
Chain Elongation ◽  
Seminiferous Tubules ◽  
Testicular Germ Cell ◽  
Abnormal Accumulation ◽  
Normal Spermatogenesis ◽  
Massive Accumulation

During normal spermatogenesis, more than half of the germ cells undergo apoptosis, but the physiological significance and molecular mechanisms of this programmed cell death are largely unknown. Because Bcl-2 functions as a death repressor, we have investigated the effect of misexpressing Bcl-2 in spermatogonia in transgenic mice using the human bcl-2 cDNA under the control of the human polypeptide chain elongation factor 1alpha (EF-1alpha) promoter. In the 2-week-old transgenic testes, exogenous Bcl-2 was expressed in spermatogonia and massive accumulation of spermatogonia was observed in seminiferous tubules by 4 weeks. At this time, only a few spermatocytes were apparent, and the accumulated cells degenerated, leading to vacuolization in some seminiferous tubules by 7 weeks. In older transgenic mice, abnormal accumulation of spermatogonia and degeneration of these germ cells was still observed, but some seminiferous tubules in which the level of Bcl-2 expression was reduced recovered normal spermatogenesis. These observations indicate that spermatogonial apoptosis is part of the normal program of mammalian spermatogenesis and is regulated by a pathway affected by Bcl-2.

scholarly journals Altered Response to Total Body Irradiation of C57BL/6-Tg (CAG-EGFP) Mice

Dose-Response ◽  
2020 ◽  
Vol 18 (3) ◽  
pp. 155932582095133
Author(s):  
Cuihua Liu ◽  
Kaoru Tanaka ◽  
Takanori Katsube ◽  
Guillaume Varès ◽  
Kouichi Maruyama ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Total Body Irradiation ◽  
Apoptotic Cell ◽  
Whole Body ◽  
Biological Research ◽  
Major Influence ◽  
Wild Type ◽  
Apoptotic Gene ◽  
Total Body

Application of green fluorescent protein (GFP) in a variety of biosystems as a unique bioindicator or biomarker has revolutionized biological research and made groundbreaking achievements, while increasing evidence has shown alterations in biological properties and physiological functions of the cells and animals overexpressing transgenic GFP. In this work, response to total body irradiation (TBI) was comparatively studied in GFP transgenic C57BL/6-Tg (CAG-EGFP) mice and C57BL/6 N wild type mice. It was demonstrated that GFP transgenic mice were more sensitive to radiation-induced bone marrow death, and no adaptive response could be induced. In the nucleated bone marrow cells of GFP transgenic mice exposed to a middle dose, there was a significant increase in both the percentage of cells expressing pro-apoptotic gene Bax and apoptotic cell death. While in wild type cells, lower expression of pro-apoptotic gene Bax and higher expression of anti-apoptotic gene Bcl-2, and significant lower induction of apoptosis were observed compared to GFP transgenic cells. Results suggest that presence of GFP could alter response to TBI at whole body, cellular and molecular levels in mice. These findings indicate that there could be a major influence on the interpretation of the results obtained in GFP transgenic mice.

Expression of bovine β-lactoglobulin transgenic mice

1999 ◽  
Vol 66 (2) ◽  
pp. 289-294 ◽  
Author(s):  
ALFONSO GUTIÉRREZ-ADÁN ◽  
ELIZABETH A. MAGA ◽  
ESMAIL BEHBOODI ◽  
JANICE S. CONRAD-BRINK ◽  
ANTHONY G. MACKINLAY ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Transgenic Mice ◽  
Transgenic Animals ◽  
Milk Composition ◽  
Developmentally Regulated ◽  
Independent Manner ◽  
Milk Protein Genes ◽  
Dairy Animals ◽  
Foreign Proteins ◽  
Β Lactoglobulin

The use of transgenic animals to manipulate milk composition has considerable potential, both for the production of biomedical proteins and for the direct manipulation of milk composition for the improvement of dairy animals and their products (for reviews, see Wall et al. 1992; Yom & Bremel, 1993). Promoters from a number of milk protein genes from a variety of species have been tested for their ability to direct the expression of foreign proteins to the mammary gland (for review, see Maga & Murray, 1995).β-Lactoglobulin (β-lg) is the major whey protein produced in ruminant milk and is part of the normal milk composition of most mammals except humans and rodents (Pervaiz & Brew, 1985). It is expressed at high levels in the mammary gland and is developmentally regulated. Transgenic mice have been produced using the complete ovine (Simons et al. 1987; Shani et al. 1992) and caprine (Ibañez et al. 1997) β-lg genes. In general, high levels of expression were obtained with the ovine β-lg gene, and expression was also seen in a position-independent manner (Whitelaw et al. 1992). Lower levels of expression were reported using the caprine β-lg gene. Here we report the production of transgenic mice using the bovine β-lg gene. We describe high expression, position-dependent, and copy number-related expression of bovine β-lg protein in the milk of six lines of transgenic mice.

scholarly journals Cre recombinase activity specific to postnatal, premeiotic male germ cells in transgenic mice

genesis ◽  
2008 ◽  
Vol 46 (12) ◽  
pp. 738-742 ◽  
Author(s):  
Patricia I. Sadate-Ngatchou ◽  
Christopher J. Payne ◽  
Andrea T. Dearth ◽  
Robert E. Braun
Keyword(s):  
Transgenic Mice ◽  
Germ Cells ◽  
Cre Recombinase ◽  
Male Germ Cells

scholarly journals 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

1999 ◽  
Vol 338 (2) ◽  
pp. 311-316 ◽  
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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