1,721,001 research outputs found
Isolated ruminant mammary stem cell population and uses thereof for production of transgenic proteins in vivo
No full textThus object of the present disclosure is to provide a bovine stem cell population able to (re)generate polarized and functional structures in vitro and in vivo when transplanted in an animal model.
A further object of the present disclosure is to provide a novel approach for transgene production in milk: given the huge proliferative and morphogenic potential of stem cells, such cells represent a preferential target for in vitro manipulation. The present disclosure concerns the use of a transgenic stem cell population that upon transplantation is able to regenerate mammary structure that secretes the transgene in their lumen.
According to the present invention said objects are achieved thanks to the solution having the characteristics referred to specifically in the ensuing claims. Thus the claims form integral part of the technical teaching herein provided in relation to the present invention.
To achieve this object, the present inventors have developed an isolated ruminant mammary stem cell population able to generate polarized and functional structures of a mammary gland tissue both in vitro and in vivo.
In an embodiment of the present disclosure the isolated ruminant mammary stem cell population is able to proliferate and differentiate into luminal and myoepithelial cells, thus generating mammary alveoli, which are able to secrete milk.
In a further embodiment of the instant invention, a transgenic isolated ruminant mammary stem cell population is provided, wherein the mammary stem cells contain within their DNA a construct encoding for at least one heterologous protein, which is secreted, preferably in the lumen of the luminal cells and/or the mammary alveoli.
In a still further embodiment the construct is a vector, being this vector selected among a lentiviral vector. Preferably the vector is provided at the N-terminal with a secretion signal and/or with a conditional promoter able to control the expression of the heterologous protein in the luminal cells and thus in milk.
In a further embodiment the heterologous protein is selected among human beta-casein, k-casein, beta-lactoglobulin, alpha-lactalbumin, monoclonal antibodies, plasma proteins such as the coagulation factors Factor XII, Factor XIII, Factor IX, albumin, human alpha 1 antitrypsin.
Further embodiments of the instant disclosure concern the use of the isolated ruminant mammary stem cell population for the regeneration of a ruminant mammary gland tissue and/or for increasing milk production in a ruminant.
In a further embodiment, the present disclosure concerns the use of the transgenic isolated ruminant mammary stem cell population for the production of the heterologous protein(s) in the milk of the ruminant, wherein the transgenic mammary stem cell population is transplanted in the mammary gland tissue of the ruminant
Correlation between estrogen plasma level and miRNAs in muscle of Piedmontese cattle
Full text linkA loss-of-function mutation of the myostatin gene has a very high prevalence in the Piedmontese cattle breed. The effect of such mutation is a double-muscle phenotype because of hypertrophy. However, differences in muscle mass development can still be detected in individuals of this breed. Such differences must be generated by other factors controlling skeletal muscle development. MicroRNAs are short noncoding RNA molecules that modulate gene expression at a post-transcriptional level. MicroRNAs have been demonstrated to be involved in skeletal muscle development, and some of them are controlled by steroid hormone signaling. Data on estrogen signaling are lacking, whereas more studies have been carried out on the effect of androgens. We aimed at identifying putative estrogen responsive miRNAs that might be involved in skeletal muscle development. At a slaughterhouse, we collected muscle samples from longissimus dorsi and blood samples. Blood 17beta-estradiol concentration was assessed, and RNA was extracted from muscle samples. The animals we sampled were divided into groups according to estrogen blood concentration, and through qPCR expression, levels of 7 muscle-related miRNAs were evaluated. We found that miR-26b (P < 0.01), miR-27a-5p (P < 0.05), miR-27b (P < 0.05), and miR-199a-3p (P < 0.01) were differentially expressed among experimental groups. Expression levels of miR-26b were reduced approximately 50% in samples with a low blood estrogen concentrations, and the other miRNAs showed a tendency to increase their expression levels when blood estrogen levels were higher. The variations of the circulating concentrations of estrogen in Piedmontese cattle might influence muscle mass development through miRNAs and thus contribute to individual variability in a breed with a high prevalence of a myostatin point mutation
Functional effect of mir-27b on myostatin expression: a relationship in Piedmontese cattle with double-muscled phenotype
No full textAbstract
Background: In Piedmontese cattle the double-muscled phenotype is an inherited condition associated to a point
mutation in the myostatin (MSTN) gene. The Piedmontese MSTN missense mutation G938A is translated to C313Y myostatin protein. This mutation alters MSTN function as a negative regulator of muscle growth, thereby inducing muscle hypertrophy. MiRNAs could play a role in skeletal muscle hypertrophy modulation by down-regulating gene
expression.
Results: After identifying a 30-UTR consensus sequence of several negative and positive modulator genes involved
in the skeletal muscle hypertrophy pathway, such as IGF1, IGF1R, PPP3CA, NFATc1, MEF2C, GSK3b, TEAD1 and
MSTN, we screened miRNAs matching to it. This analysis led to the identification of miR-27b, miR-132, miR-186 and
miR-199b-5p as possible candidates. We collected samples of longissimus thoracis from twenty Piedmontese and
twenty Friesian male bovines. In Piedmontese group miR-27b was up-regulated 7.4-fold (p < 0.05). Further, we
report that the level of MSTN mRNA was about 5-fold lower in Piedmontese cattle vs Friesian cattle (p < 0.0001)
and that less mature MSTN protein was detected in the Piedmontese one (p < 0.0001). Cotransfection of miR-27b
and psi-check2 vector with the luciferase reporter gene linked to the bovine wild-type 30-UTR of MSTN strongly
inhibited the luciferase activity (79%, p < 0.0001).
Conclusions: These data demonstrate that bovine MSTN is a specific target of miR-27b and that miRNAs contribute
to explain additive phenotypic hypertrophy in Piedmontese cattle selected for the MSTN gene mutation, possibly
outlining a more precise genetic signature able to elucidate differences in muscle conformation
Purification of ruminant mammary stem cell population and uses thereof for production of transgenic proteins in vivo
No full text
A Sorting Strategy For Bovine Mammary Progenitors
No full textThe mammary gland is a bilayered system organized into a series of branching ducts that terminate in secretory alveoli. Their epithelium is composed of an inner layer of cytokeratin(CK)18+ luminal cells, which secrete milk, and an outer layer of contractile CK14+ myoepithelial cells, which contribute to milk ejection during lactation. The existence of mammary renewable stem cells (MaSCs) has been deduced from the profound regenerative ability of the mammary epithelium. It undergoes different morphogenetic changes during puberty and during the subsequent pregnancies. The knowledge of MaSCs properties and functions should represent an interesting opportunity in the field of animal production as the appropriate regulation of these cells should benefit milk yeld. The aim of our study is to characterize the MaSCs population of bovine species and set up a sorting strategy to purify different primitive populations. The Colony-Forming Cell (CFC) assay performed on bovine mammary samples obtained from virgin, two years old cows revealed the presence in the mammary gland of both myoepithelial and luminal-restricted progenitor cells. In order to select and isolate the different types of progenitors we have performed a flow cytometric analysis of cells dissociated from bovine mammary tissue. Cells were then stained with antibodies directed to CD49f, whose expression at high levels is associated with progenitor and stem cell activity, and p-cadherin, that is normally expressed in the myoepithelial/basal layer of the mammary epithelium. After depletion of hematopoietic cells (using an anti-CD45 antibody) we identified four different subpopulations. They were characterized by different levels of CD49f/P-cad expression: CD49f/P-cad- (15,6%), CD49f++/P-cad- (25,4%), CD49f+/P-cad+ (27,1%), CD49f+/P-cad++ (10,2%). After sorting, the CFC assay performed with the cells of the different subpopulations showed that CD49f++/P-cad- population was able to generate colonies with an higher frequency compared to the others, revealing an enrichment in stem cells. In particular, 35,7% of colonies showed a myoepithelial phenotype and 64,3% a luminal one. In order to analyze whether these cells also had an in vivo tissue regenerative activity, the four subpopulation were implanted in collagen gels under the kidney capsule of NOD/SCID immunodeficient mice in numbers proportional to their frequency in the CD45- population. Immunohistochemical analysis of gels recovered 4 weeks later revealed that only CD49f++/P-cad- subpopulation was able to regenerate in vivo mammary alveolar structures characterized by a lumen surrounded by two layers of cells: an inner K18+ layer and an outer K14+ layer. The CFC assay performed with single cell suspension obtained from recovered gels showed a higher colony-forming ability of CD49f++/P-cad- population compared to the others. Therefore the structures produced in these gels contained clonogenic progenitors, that were able to form the same types of colonies observed from freshly isolated cells (36,2% of myoepithelial colonies and 63,8% of luminal ones). By using a novel sorting strategy we show that the mammary myoepithelial cell layer can be divided in different subpopulations. Each one has a different progenitor content and only in one fraction (CD49f++/P-Cad-) we were able to detect a subset of adult stem cells able to regenerate a mammary epithelium. This sorting strategy might prove useful for understanding the stem cells population dynamics during the different phases that the mammary gland undergoes. Besides, given the similar organization of the mammary tissue in the bovine and the human species, the cow might represent a much better animal model than the mouse
Identification of goat mammary stem/progenitor cells
No full textGoat mammary gland epithelial cells have been used to establish primary and permanent cell lines, but to date, no data have been available regarding mammary stem cells (MaSCs) in this species. The detection and characterization of goat MaSCs is an important task for a better understanding of the cyclic character of mammary gland development, which will also offer the potential for manipulation of lactation yield and persistency. The objective of the present study was to demonstrate that a subpopulation of goat MaSCs resides in the goat mammary gland. Mammary tissue from lactating Saanen goats (Capra hircus) was dissociated and processed to a single-cell suspension. Using an in vitro colony-forming assay, we demonstrated that distinct colony types, which expressed specific lineage markers, arose from unipotent progenitors. Using two different growth media, we showed that the frequencies of caprine clonogenic progenitors differed according to growth conditions. Goat epithelial cells were transplanted under the kidney capsule of nonobese diabetic/severe combined immunodeficient (NOD/ SCID) mice, where they formed organized, bilayered structures. Our results indicate the presence of goat MaSCs in the caprine mammary gland. To our knowledge, these data represent the first description of the tissue hierarchy of the goat mammary gland and demonstrate the regenerative potential of adult goat MaSCs. © 2012 by the Society for the Study of Reproduction, Inc
- …
