scholarly journals Foreign Gene Expression in the Mouse Cauda Epididymis is Regulated by Androgens

2018 ◽  
Vol 2 (4) ◽  
pp. 671-678
Author(s):  
Pedro Esponda
Keyword(s):  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Foreign Gene ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

scholarly journals Senile Octodon degus Reproductive Strategy: Morphometric and Histological Description of the Penis

2018 ◽  
Vol 2 (4) ◽  
pp. 679-688
Author(s):  
María Reyes ◽  
Eduardo Bustos-Obregón ◽  
Mariana Rojas
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Female Reproductive Tract ◽  
Total Period ◽  
Green Fluorescent ◽  
In Vivo Gene Transfer

This paper deals with the efficiency of in vivo gene transfer to the mouse cauda epididymis and its relation to androgens. Previous experiments in the female reproductive tract have indicated that the efficiency of transfection is related to the hormonal stage of the animal, nevertheless no analysis have been done in the male tract. We used in vivo gene transfer to the mouse cauda epididymis employing a gene construction that expresses the Green Fluorescent Protein (GFP). Untreated and Testosterone treated males were employed. Testosterone injections (5 μg/g weight) were done from 2 days before the gene transfer, and treatment continued each day during a total period of 15 days. Fluorescence microscopy observations showed the expression of GFP in the cytoplasm of the principal cells in the epididymal tubules. The application of the QWin Program that measures the percentage of fluorescent areas showed that they are increased in the epididymis of treated males. This increase was particularly observed two days after gene injections (from 32.24 % in untreated animals to 47.62 % in testosterone treated males) and after seven days (from 29.98 % to 43.05 %). The possibility to improve transfection efficiency would increase the knowledge on epididymal physiology and would permit to modify the fertilizing capacity in mammals.

Phenotypic and immunologic factors affecting plasmid-mediated in vivo gene transfer to rat diaphragm

1996 ◽  
Vol 270 (6) ◽  
pp. L1023-L1030 ◽  
Author(s):  
B. J. Petrof ◽  
G. Acsadi ◽  
J. Bourdon ◽  
N. Matusiewicz ◽  
L. Yang
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Reporter Gene ◽  
Transfection Efficiency ◽  
Foreign Gene ◽  
Rat Diaphragm ◽  
Foreign Gene Expression ◽  
Hindlimb Muscles ◽  
In Vivo Gene Transfer

Little is known about the molecular mechanisms governing adaptive responses of the diaphragm in the setting of lung disease. By permitting the study of regulatory elements and the effects of overexpressing genes of interest, direct in vivo gene transfer to the diaphragm could be used as a tool to address such questions. Therefore, we evaluated parameters affecting transfection efficiency and duration of foreign gene expression in the diaphragm after plasmid-mediated gene transfer. Reporter gene constructs were injected into adult rat diaphragm and hindlimb muscles. Transfection efficiency at 8-10 days postinjection was decreased in large caliber ( > 1,000 microns2) and type II myosin heavy chain (MHC)-expressing fibers. There were also strong trends toward augmented transfection efficiency in type I MHC- and embryonic MHC-expressing fibers. All diaphragms demonstrated evidence of muscle injury and inflammatory cell infiltrates at this early time point. By 30 days postinjection, however, neither inflammation nor reporter gene expression was detectable in diaphragm or hindlimb muscles of immunocompetent animals. By contrast, immunosuppressed rats (given cyclosporine; 15 mg.kg-1. day-1) showed high levels of foreign gene expression at 30 days postinjection, which remained stable up to 60 days. Therefore, exploitation of plasmid-mediated in vivo gene transfer as a tool for studying regulated gene expression in the diaphragm may be facilitated by the use of immunodeficient animal models.

scholarly journals Redistribution of the intra-acrosomal EGFP before acrosomal exocytosis in mouse spermatozoa

Reproduction ◽  
2015 ◽  
Vol 149 (6) ◽  
pp. 657-663 ◽  
Author(s):  
Noritaka Hirohashi ◽  
Florenza A La Spina ◽  
Ana Romarowski ◽  
Mariano G Buffone
Keyword(s):  
Acrosome Reaction ◽  
Reproductive Tract ◽  
Fluorescent Protein ◽  
Female Reproductive Tract ◽  
Morphological Alterations ◽  
Acrosomal Exocytosis ◽  
Green Fluorescent ◽  
Mammalian Spermatozoa

Mammalian spermatozoa must undergo complex physiological and morphological alterations within the female reproductive tract before they become fertilization competent. Two important alterations are capacitation and the acrosome reaction (AR), by which spermatozoa become capable of penetrating the zona pellucida (ZP) of the oocyte. Although various biochemical stimulants have been reported to induce the AR, the true physiological inducer in vivo remains to be identified. Previously, it has been reported that most fertilizing spermatozoa undergo the AR before contacting the ZP and that only a small fraction of in vitro-capacitated spermatozoa can penetrate the ZP. Therefore, it is important to identify which capacitating spermatozoa undergo the AR in response to potential AR inducers such as progesterone. Here we show that spermatozoa undergo a dynamic rearrangement of the acrosome during in vitro capacitation. This involves the rapid movement of an artificially introduced soluble component of the acrosome, enhanced green fluorescent protein (EGFP), from the acrosomal cap region to the equatorial segment (EQ) of the sperm head. Spermatozoa exhibiting the EQ pattern were more sensitive to progesterone than were those without it. We suggest that spermatozoa that are ready to undergo acrosomal exocytosis can be detected by real-time EGFP imaging. This offers a promising new method for identifying where spermatozoa undergo the AR in the female reproductive tract in vivo.

scholarly journals Retroviral-Mediated Transfer of the Green Fluorescent Protein Gene Into Murine Hematopoietic Cells Facilitates Scoring and Selection of Transduced Progenitors In Vitro and Identification of Genetically Modified Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (5) ◽  
pp. 1777-1786 ◽  
Author(s):  
Derek A. Persons ◽  
James A. Allay ◽  
Esther R. Allay ◽  
Richard J. Smeyne ◽  
Richard A. Ashmun ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Fluorescent Protein ◽  
High Titer ◽  
Hematopoietic Cells ◽  
Green Fluorescent Protein Gene ◽  
Retroviral Gene Transfer ◽  
Green Fluorescent ◽  
Marrow Cells

Abstract We have investigated the utility of the green fluorescent protein (GFP) to serve as a marker to assess retroviral gene transfer into hematopoietic cells and as a tool to identify and enrich for cells expressing high levels of the vector-encoded transcript. GFP, by virtue of a naturally occurring chromophore encoded in its primary sequence, displays autonomous fluorescence, thus eliminating the need for antibody or cytochemical staining to detect its expression. A bicistronic murine stem cell virus (MSCV)-based retroviral vector was constructed containing the GFP cDNA and a mutant, human dihydrofolate reductase gene. High-titer, ecotropic retroviral producer cells free of replication competent virus were generated and used to transduce murine bone marrow cells by cocultivation. Within 24 hours after completion of the transduction procedure, a high proportion (40% to 70%) of the marrow cells were intensely fluorescent compared to mock-transduced cells or cells transduced with a control retrovirus. Erythroid and myeloid hematopoietic colonies derived from GFP-transduced marrow were easily scored for retroviral gene transfer by direct in situ fluorescence microscopy. Clonogenic progenitors expressing increased levels of antifolate drug resistance could be enriched from the GFP-transduced marrow population by fluorescence activated cell sorting of cells expressing high levels of GFP. In vivo, splenic hematopoietic colonies and peripheral blood cells from animals transplanted with GFP-transduced marrow displayed intense fluorescence. These results show that GFP is an excellent marker for scoring and tracking gene-modified hematopoietic cells and for allowing rapid selection and enrichment of transduced cells expressing high levels of the transgene.

scholarly journals Transfer of Green Fluorescent Protein (GFP) Gene to Betta splendens Embryos by Transfection and Electroporation Methods

Omni-Akuatika ◽  
2018 ◽  
Vol 14 (2) ◽  
Author(s):  
Eni Kusrini ◽  
Alimuddin Alimuddin ◽  
Erma Primanita Hayuningtyas ◽  
Syuhada Restu Danupratama
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Betta Splendens ◽  
Foreign Gene ◽  
Gfp Expression ◽  
Dna Concentration ◽  
Gfp Gene ◽  
Green Fluorescent

Transfection and electroporation method shave a high possibility to apply towards transgenic production of small eggs size fish species.  This study aimed to examine the potential of transfection and electroporation methods to use for transferring a foreign gene into betta fish (Betta splendens) embryos using green fluorescent protein (GFP) gene as a model.  Fish were spawned naturally in the ratio of male: female was 1:1, then a total of 200 eggs were taken for each treatment.  Transfection was performed for 30 minutes (room temperature of about 25 °C) at two-cell stage of embryos using transfast reagent.  Transfection reaction consisted of 0.75 µL transfast reagent, 0.25 µL GFP expression vector (DNA concentration: 50 µg/µL) and 99 µL NaCl solution (concentration: 0,95%).  Electroporation was performed using 125 volt cm-1, 3 times pulse frequency at one second interval and pulse length of 7 micro seconds.  A volume of 800 µL GFP expression vector solution (DNA concentration: 50 µg/ µL) in PBS was used for electroporation.  The successful of foreign gene transfer was determined by PCR method with GFP specific primers.  The results showed that hatching rate of eggs in transfection treatment was 67.08%, while the electroporation was 72.09%.  Survival of larvae in transfection treatment was 73.00%, while the electroporation was 75.00%.  The results of PCR analysis showed that transfection method allowed 65% of the survived fish carrying GFP gene, whereas the electroporation method was 70%.  Thus, foreign gene transfer in betta fish can be conducted using the transfection and electroporation methods. 

scholarly journals Adenoviral Gene Transfer of a u-PA Receptor-binding Plasmin Inhibitor and Green Fluorescent Protein: Inhibition of Migration and Visualization of Expression

2000 ◽  
Vol 84 (09) ◽  
pp. 460-467 ◽  
Author(s):  
M. L. M. Lamfers ◽  
M. J. Wijnberg ◽  
J. M. Grimbergen ◽  
L. G. M. Huisman ◽  
M. C. Aalders ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Cell Migration ◽  
Green Fluorescent Protein ◽  
Gene Transfer ◽  
Receptor Binding ◽  
Fluorescent Protein ◽  
Transfection Efficiency ◽  
Adenoviral Gene Transfer ◽  
Amino Terminal ◽  
Green Fluorescent

SummarySmooth muscle cell migration plays a role in the development of intimal hyperplasia. Given the established role of the plasminogen activation system in cell migration, an approach to therapy is to overexpress an inhibitor of plasmin. Therefore, an adenoviral vector was constructed encoding the hybrid protein ATF.BPTI, which contains the active domain of bovine pancreas trypsin inhibitor (BPTI), fused to ATF, the amino terminal fragment or receptor-binding domain of u-PA. Adenoviral vectors expressing ATF and BPTI individually were also constructed, and a fourth vector was constructed encoding ATF.BPTI linked by an internal ribosomal entry site to Green Fluorescent Protein (ABIG). Both the expression and functionality of the recombinant proteins were established in human vascular smooth muscle cells. Adenoviral gene transfer of ATF.BPTI inhibited SMC migration more efficiently than the expression of ATF or BPTI individually. Expression of ABIG resulted in the co-expression of ATF.BPTI and Green Fluorescent Protein, thereby providing a tool to monitor transfection efficiency and the behavior of the transfected cells.

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