scholarly journals Expression analysis of the individual bovine β-,αs2- and κ-casein genes in transgenic mice

1995 ◽  
Vol 311 (3) ◽  
pp. 929-937 ◽  
Author(s):  
M Rijnkels ◽  
P M Kooiman ◽  
P J A Krimpenfort ◽  
H A de Boer ◽  
F R Pieper
Keyword(s):  
Mammary Gland ◽  
Transgenic Mice ◽  
Bovine Milk ◽  
Regulatory Elements ◽  
Regulation Of Expression ◽  
Casein Gene ◽  
Expression Levels ◽  
Beta Casein ◽  
Casein Genes ◽  
High Level

To identify cis-acting regulatory elements involved in the regulation of expression of the casein genes, the bovine beta-, alpha s2- and kappa-casein genes were isolated from cosmid libraries and introduced into the murine germline. Bovine casein expression was analysed at the RNA and protein level. The bovine beta-casein gene, including 16 kb of 5′- and 8 kb of 3′-flanking region, appeared to be expressed in all 12 transgenic mouse lines analysed. In 50% of these lines expression levels in milk exceeded 1 mg/ml. Three lines displayed expression levels comparable with or well above (20 mg/ml) the beta-casein levels in bovine milk. Transgene expression was restricted to the mammary gland. Strong induction of expression occurred at parturition and thus resembled the bovine rather than the murine pattern. In spite of this high-level tissue-specific and developmentally regulated expression, beta-casein expression levels were integration-site-dependent, suggesting that not all elements involved in regulation of expression were included in this beta-casein clone. Neither the bovine alpha s2- nor the kappa-casein gene, including 8 kb and 5 kb of 5′- and 1.5 kb and 19 kb of 3′-flanking sequences respectively, were properly expressed in transgenic mice. However, they were transcribed in stably transfected mouse mammary epithelial cells. This indicates that regulatory elements required for high-level, mammary gland-specific expression are not present in the alpha s2- and kappa-casein clones used in this study and are probably located elsewhere in the casein gene locus.

Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice

1989 ◽  
Vol 9 (2) ◽  
pp. 560-565
Author(s):  
K F Lee ◽  
S H Atiee ◽  
J M Rosen
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Transgenic Mice ◽  
Fusion Gene ◽  
Chloramphenicol Acetyltransferase ◽  
Fusion Genes ◽  
Specific Expression ◽  
Casein Gene ◽  
Noncoding Exon ◽  
Beta Casein

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.

scholarly journals Differential regulation of rat beta-casein-chloramphenicol acetyltransferase fusion gene expression in transgenic mice.

1989 ◽  
Vol 9 (2) ◽  
pp. 560-565 ◽  
Author(s):  
K F Lee ◽  
S H Atiee ◽  
J M Rosen
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Transgenic Mice ◽  
Fusion Gene ◽  
Chloramphenicol Acetyltransferase ◽  
Fusion Genes ◽  
Specific Expression ◽  
Casein Gene ◽  
Noncoding Exon ◽  
Beta Casein

Previous studies in our laboratory have demonstrated the mammary-specific expression of the entire rat beta-casein gene with 3.5 kilobases (kb) of 5' and 3.0 kb of 3' DNA in transgenic mice (Lee et al., Nucleic Acids Res. 16:1027-1041, 1988). In an attempt to localize sequences that dictate this specificity, lines of transgenic mice carrying two different rat beta-casein promoter-bacterial chloramphenicol acetyltransferase (cat) fusion genes have been established. Twenty and eight lines of transgenic mice carrying two fusion genes containing either 2.3 or 0.5 kb, respectively, of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A were identified, most of which transmitted the transgenes to their offspring in a Mendelian pattern. CAT activity was detected predominantly in the lactating mammary gland of female transgenic mice but not in the male mammary fat pad. A several-hundred-fold variation in the level of cat expression was observed in the mammary gland of different lines of mice, presumably due to the site of integration of the transgenes. CAT activity was increased in the mammary gland during development from virgin to midpregnancy and lactation. Unexpectedly, the casein-cat transgenes were also expressed in the thymus of different lines of both male and female mice, in some cases at levels equivalent to those observed in the mammary gland, and in contrast to the mammary gland, CAT activity was decreased during pregnancy and lactation in the thymus. Thus, 0.5 kb of 5'-flanking DNA of the rat beta-casein gene along with noncoding exon I and 0.5 kb of intron A are sufficient to target bacterial cat gene expression to the mammary gland of lactating mice.

scholarly journals The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽  
1995 ◽  
Vol 85 (2) ◽  
pp. 319-329 ◽  
Author(s):  
S Dziennis ◽  
RA Van Etten ◽  
HL Pahl ◽  
DL Morris ◽  
TL Rothstein ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Heterologous Gene Expression ◽  
Myeloid Cells ◽  
Reporter Genes ◽  
Regulatory Elements ◽  
Specific Expression ◽  
High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

Distal regulatory elements from the mouse metallothionein locus stimulate gene expression in transgenic mice

1993 ◽  
Vol 13 (9) ◽  
pp. 5266-5275
Author(s):  
R D Palmiter ◽  
E P Sandgren ◽  
D M Koeller ◽  
R L Brinster
Keyword(s):  
Transgenic Mice ◽  
Regulatory Elements ◽  
High Level Expression ◽  
Hypersensitive Sites ◽  
Enhanced Expression ◽  
Rat Growth ◽  
Flanking Sequences ◽  
High Level ◽  
Distal Regulatory Elements ◽  
Flanking Regions

DNA regions of 10 and 7 kb that flank the mouse metallothionein II (MT-II) and MT-I genes, respectively, were combined with a minimally marked MT-I (MT-I*) gene and tested in transgenic mice. This construct resulted in (i) position-independent expression of MT-I* mRNA and copy number-dependent expression, (ii) levels of hepatic MT-I mRNA per cell per transgene that were about half that derived from endogenous MT-I genes, (iii) appropriate regulation by metals and hormones, and (iv) tissue distribution of transgene mRNA that resembled that of endogenous MT-I mRNA. These features were not observed when MT-I* was tested without the flanking regions. These MT-I flanking sequences also improved the expression of rat growth hormone reporter genes, with or without introns, that were under the control of the MT-I promoter. Moreover, they enhanced expression from two of four heterologous promoters/enhancers that were tested. Deletion analysis indicated that regions known to have DNase I-hypersensitive sites were necessary but not sufficient for high-level expression. These data suggest that the DNA regions flanking the mouse MT-I and MT-II genes have functions like the locus control regions described for other genes.

scholarly journals Assessment of Biological Potential Realization in Cattle of the Kostroma Breed with Different Allelic Variants of Beta-Casein Gene

2021 ◽  
pp. 22-28
Author(s):  
А.А. Чаицкий ◽  
Н.С. Баранова
Keyword(s):  
Genetic Mutation ◽  
Biological Efficiency ◽  
Allelic Variants ◽  
Casein Gene ◽  
Biological Potential ◽  
Beta Casein ◽  
Casein Protein ◽  
High Level ◽  
Relationship Of ◽  
Quantitative Indicators

Среди сложных белков молока – казеинов – чаще всего встречаются α- и β-казеин. Ген бета-казеина имеет 12 аллельных вариантов, среди которых получили преимущество варианты А1 и А2. При этом, А2 – естественная для человеческого организма разновидность белка бета-казеина, а тип А1 возник в результате природной генетической мутации. Для получения максимального объёма продукции лучшего качества при переориентации производства на молоко А2 необходимо учитывать как основные количественные показатели продуктивности крупного рогатого скота, так и коэффициенты, определяющие эффективность использования коров с точки зрения биологической полноценности продукции, такие как биологической эффективности коров (БЭК), биологической полноценности молока (КБП), кормового коэффициента (FCR) и валовой эффективности использования кормов (GFE). Представлено сравнение количественных и качественных показателей животных разного возраста и групп с учётом степени кровности по улучшающей породе, а также с учётом генотипа по бета-казеину. Впервые проведён анализ реализации биологического потенциала крупного рогатого скота костромской породы с разными аллельными вариантами гена бета-казеина. В ходе исследования установлено, что животные генотипа А2А2 по бета-казеину отличались высоким уровнем БЭК и КБП, а также характеризовались достоверно более высоким уровнем реализации сухого вещества корма в молоко (FCR, GFE), чем у животных А1А2. Так, удои у коров с генотипом А2А2 с кровностью 50% по улучшающей породе за полновозрастную лактацию на 2514 кг больше, чем у животных генотипа А1А2, а коэффициенты БЭК, КБП и FCRвыше на 39,79%, 15,81% и 0,57 кг соответственно. Among the complex proteins of milk – caseins – most often there are α- and β-casein. The beta-casein gene has 12 allelic variants, among which the A1 and A2 variants have gained advantage. At the same time A2 is a natural variety of beta-casein protein for the human body and type A1 arose as a result of a natural genetic mutation. To obtain the maximum volume of production of better quality when reorienting production to milk A2 it is necessary to take into account both the main quantitative indicators of cattle productivity and the coefficients that determine the efficiency of cows use in terms of biological usefulness of products, such as the biological efficiency of cows (BEC), the biological usefulness of milk (BUC), feed coefficient rate (FCR) and gross feed efficiency (GFE). A comparison of quantitative and qualitative indicators of animals of different ages and groups is presented, taking into account the degree of blood relationship by improving breed, as well as taking into account the genotype by beta-casein. For the first time an analysis of the implementation of the biological potential of cattle of the Kostroma breed with different allelic variants of the beta-casein gene was carried out. During the research it was established that animals of a genotype A2A2 on beta casein differed in the BEC and BUC high level and also were characterized by significantly higher level of implementation of dry matter of feed into milk (FCR, GFE), than at animals A1A2. Thus, yields in cows with a genotype A2A2 with a blood relationship of 50% for the improving breed for full-age lactation 2514 kg more than in animals of the genotype A1A2 and the coefficients of BEC, BUC and FCR are higher by 39.79%, 15.81% and 0.57 kg, respectively. Keywords:

scholarly journals Sequence Characterisation and Genotyping of Allelic Variants of Beta Casein Gene Establishes Native Cattle of Ladakh to be a Natural Resource for A2 Milk

2018 ◽  
Vol 3 (2) ◽  
pp. 177
Author(s):  
Monika Sodhi ◽  
R. S. Kataria ◽  
Saket K. Niranjan ◽  
Parvesh K. ◽  
Preeti Verma ◽  
...  
Keyword(s):  
Natural Resource ◽  
Bovine Milk ◽  
Human Consumption ◽  
Coding Region ◽  
Low Oxygen ◽  
Casein Gene ◽  
Allelic Distribution ◽  
Oxygen Condition ◽  
Beta Casein ◽  
Native Cattle

Bovine milk is regarded as nature's perfect food due to presence of vital nutrients. However some peptides are generated after proteolytic digestion of β-casein that have opioid properties and may increase the risk of chronic diseases. There are 13 genetic variants of bovine beta-casein; out of these A1 and A2 are the most common in dairy cattle breeds. The A1 and A2 variants differ only at position 67, which is histidine in A1 or proline in A2 milk. Earlier published reports have indicated that A1 β casein could be responsible for several health disorders like diabetes, coronary heart disease etc. while A2 β-casein is generally considered safe for human consumption. In the present study, an effort was made to sequence characterize β casein gene and identify allelic distribution of A1A2 alleles in native cattle of Ladakh region adapted to high altitude and low oxygen condition. The data showed 2 non-synonymous variations in coding region, while 5’UTR was completely conserved. The 3’UTR showed 2 more variations in Ladakhi samples. Further, the genotyping in 85 Ladakhi cattle for A1A2 alleles revealed that in Ladakhi cattle, A2 allele is predominantly present as reported for some of the other Indian breeds. The frequency of A2 allele was 0.90 and frequency of A2A2 genotype was found to be 0.79 in Ladakhi cattle. The present data strongly indicate that local cattle of Ladakh with higher frequency of A2 allele and A2A2 genotype is natural resource for A2 milk.  Systematic efforts should be made for long term conservation and genetic improvement of this invaluable genetic resource of Ladakh.

scholarly journals beta-Casein mRNA sequesters a single-stranded nucleic acid-binding protein which negatively regulates the beta-casein gene promoter.

1994 ◽  
Vol 14 (9) ◽  
pp. 6004-6012 ◽  
Author(s):  
S Altiok ◽  
B Groner
Keyword(s):  
Epithelial Cells ◽  
Untranslated Region ◽  
Mammary Epithelial Cells ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Hormonal Control ◽  
Mammary Epithelial ◽  
Casein Gene ◽  
Lactogenic Hormone ◽  
Beta Casein

beta-Casein gene expression in mammary epithelial cells is under the control of the lactogenic hormones, glucocorticoids, insulin, and prolactin. The hormonal control affects gene transcription, and several regulatory elements in the beta-casein gene promoter between positions -80 and -221 have previously been identified. A region located in the promoter between positions -170 and -221 contains overlapping sequences for negative and positive regulatory elements. A sequence-specific single-stranded DNA-binding factor (STR), composed of two proteins with molecular masses of 35 and 54 kDa, recognizes the upper strand of this region and has a repressing role in transcription. High-level STR binding activity was observed in nuclear extracts from mammary glands of pregnant and postlactating mice and from noninduced HC11 mammary epithelial cells, cells with a low level of transcriptional activity of the beta-casein gene. STR activity is downregulated in mammary epithelial cells during lactation of the animals and after lactogenic hormone induction of HC11 cells in culture. These cells strongly transcribe the beta-casein gene. We investigated the mechanism of downregulation and found that a lactogenic-hormone-induced molecule (I-STR) inhibits STR from binding to its DNA target. I-STR is composed of RNA. STR is sequestered into the cytoplasm by I-STR after lactogenic hormone induction of mammary epithelial cells and remains present in an RNA-bound form. A high-affinity STR binding site was found in the 5' untranslated region of beta-casein mRNA. We propose that beta-casein mRNA can function as I-STR. beta-Casein mRNA may positively regulate its own transcription by translocating STR from the nucleus to the cytoplasm. The beta-casein STR binding sequence increases expression of a transfected beta-galactosidase gene when it is placed into the 5' untranslated region sequence of the mRNA. STR may have a positive role in posttranscriptional regulation.

Functional study of the equine β-casein and κ-casein gene promoters

2005 ◽  
Vol 72 (S1) ◽  
pp. 34-43 ◽  
Author(s):  
Tina Lenasi ◽  
Nadja Kokalj-Vokac ◽  
Mojca Narat ◽  
Antonella Baldi ◽  
Peter Dovc
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Reporter Gene ◽  
Gene Promoter ◽  
Reporter Gene Expression ◽  
Proximal Promoter ◽  
Gene Promoters ◽  
Tissue Specific ◽  
Casein Gene ◽  
Casein Genes

Casein genes are expressed in a tissue-specific and highly coordinated manner. The main goals of casein gene promoter studies are to unravel cis- and trans-acting factors involved in the complex signalling pathway controlling milk production, and to explore the possibility of using these promoters for tissue-specific production of heterologous proteins in the mammary gland. Here we present a comparative study of the equine β-casein and κ-casein gene proximal promoters. In order to confirm the assumption that in the horse, as in other mammalian species, casein genes are organized in a cluster located on a single chromosome, we performed in situ hybridization of pro-metaphase chromosomes with two BAC clones containing different equine casein genes. Sequence analysis of the β-casein and κ-casein gene proximal promoters revealed binding sites for activators (STAT5, GRE, NF1, MAF) and repressors (YY1, PMF), characteristic for casein genes. The alignments of casein gene promoters revealed the highest sequence identity in the proximal promoter region between the equine and human β-casein gene promoters. We directly compared the activity of equine β-casein and κ-casein gene promoters in vitro using bovine mammary gland cell line BME-UV1. In this system, the κ-casein gene proximal promoter activated the reporter gene expression more efficiently than the β-casein gene promoter of approximately the same length. The 810 bp of β-casein promoter activated the reporter gene expression more efficiently than the long fragment (1920 bp) and the 1206 bp fragment of the same promoter, which included also 396 bp of 5′ UTR.

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