BLG‐e1 – A novel regulatory element in the distal region of the β‐lactoglobulin gene promoter

Reichenstein, Moshe; German, Tania; Barash, Itamar

doi:10.1016/j.febslet.2005.02.061

Cited by 4 publications

(4 citation statements)

References 43 publications

(53 reference statements)

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“…The promoter of the milk protein gene (ovine or caprine beta-lactoglobulin) has been used to target high level expression of foreign protein in the milk of transgenic mice [20–23]. In our previous study, the strategy of a BLG/CMV based vector efficiently drove the human lactoferrin cDNA gene to express in the milk of transgenic mice [7].…”

Section: Discussionmentioning

confidence: 99%

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

Yuan

et al. 2014

Journal of Analytical Methods in Chemistry

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To improve nutrient content of goat milk, we describe the construction of a vector (pBLAC) containing a hybrid goat β-lactoglobulin (BLG) promoter/cytomegalovirus (CMV) enhancer. We also describe the generation of transgenic goats expressing rhLA by somatic cell nuclear transfer (SCNT). Of 334 one-cell stage embryos derived from three transgenic cell lines and 99 embryos derived from non-transgenic (NT) cells surgically transferred to the oviducts of 37 recipients, two recipients delivered two kids (2%) from the non-transfected line and five recipients delivered six kids (1.8%) from transgenic cell lines, three of which died within 2 days. Compared to the NT donor cells, transfection of donor cells does not negatively affect the development of nuclear transfer embryos into viable transgenic offspring. However, the clone efficiency in cell line number 1 was lower than that in numbers 2 and 3, and in the NT lines (0.9% versus 1.9% 2.4% and 2%; P < 0.05). Two transgenic cloned goats expressed rhLA in the milk at 0.1–0.9 mg/mL. The mammary gland-specific expression vector pBLAC with hybrid BLG/CMV can drive the hLA gene to express in vitro and in vivo. These data establish the basis for use of a hybrid promoter/enhancer strategy to produce rhLA transgenic goats.

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Section: Discussionmentioning

confidence: 99%

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

Yuan

et al. 2014

Journal of Analytical Methods in Chemistry

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“…Briefly, tissue pieces were washed, collected and sonicated on ice (SONICS Vibra cell, Newtown, CT) in 500 μl SDS lysis buffer. DNA was sheared to lengths of between 200 and 1,000 bp and ChIP was performed with anti‐acetyl histone H4 polyclonal antibodies (Upstate) diluted 1:200 as previously described 40. DNA‐protein crosslinking was reversed at 65°C for 4 hr in the presence of 5 M NaCl and DNA was recovered by phenol/chloroform extraction and ethanol precipitation.…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was isolated from the inguinal and thoracic mammary glands with TRIZOL (Gibco/BRL, Gaithersburg, MD) as previously described. 40 DNase-I-treated aliquots (0.2 lg) were reverse-transcribed using a superscript II reverse transcriptase (RT) kit and oligo(dT) primers (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol.…”

Section: Rna Extraction and Reverse Transcriptionmentioning

confidence: 99%

Tumors caused by overexpression and forced activation of Stat5 in mammary epithelial cells of transgenic mice are parity‐dependent and developed in aged, postestropausal females

Eilon

Groner

Barash

2007

Intl Journal of Cancer

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In transgenic mice overexpressing Stat5 or a constitutively activated Stat5 variant (STAT5ca), we show for the first time that parity is required for the development of tumors in postestropausal females. Tumors were detected in glands of multiparous transgenic female mice after latency period of 14 months, but rarely in their age-matched virgin (AMV) counterparts. This period was not affected by distinguishable tumor pathologies and was not dependent upon transgenic Stat5 variant. To associate Stat5 deregulation, parity and the postestropausal tumor occurrence with mammary cancer formation, the activities of endogenous and transgenic Stat5 were measured in the glands of aged multiparous and AMV females. No differences in phosphorylated Stat5 (pStat5) levels were found between the 2 cohorts. However, promoter sequences comprising the Stat5 binding sites from the cyclin D1 or the bcl-x genes associate differentially with acetylated histone H4 in aged multiparous and AMV STAT5ca transgenic females. Individual epithelial cells varied greatly with respect to the presence of nuclear pStat5. A small subset of epithelial cells, in which pStat5 and cyclin D1 were co-expressed, was exclusively present in the multiparous glands. Changes in chromatin structure might persist past the reproductive life time of the multiparous mice and contribute to the transcription of the cyclin D1 gene by activated Stat5. This may cause the detectable expression of cyclin D1 and add to the process of tumorigenesis. ' 2007 Wiley-Liss, Inc.Key words: transgenic mice; mammary gland; breast; Stat5; cancer; parity Epidemiological studies have established a correlation between parity and the incidence of breast cancer in women.1-3 Parity has a protective effect, which depends on an early age of full-term pregnancy and is augmented by the total number of pregnancies. 4,5 In contrast, first pregnancy in women at their 30s is considered a life time risk factor. 2,6 A transient increase in risk for breast cancer after delivery has also been found and augmented in women at their 30s. 7,8 The diversity between the protective and inductive effects of parity on breast cancer was mainly associated with the be-directional double edged action of sex steroid hormones. Estrogen and progesterone could induce breast cancer by growth promoting effect on premalignant or preexisting malignant cells and protect from the disease by exerting persistent changes during a critical period of adolescence with long terms effects on cell proliferation and stem cells population.9-14 The detailed mechanisms regulating the action of sex steroid hormones remain elusive, but most likely involve signaling pathways that govern the balance between apoptosis, proliferation and differentiation. Recently, the mammary microenvironment was also associated with the transient increase in breast cancer risk. It was proposed that remodeling of the mammary gland during involution induces proinflammatory and wound-healing signals that are pro-oncogenic and contribute to cancer promotion and p...

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“…Bioactive foreign proteins have been expressed specifically in the mammary gland under the direction of specific milk regulatory sequences (Liu et al, 2012). These include sequences obtained from goat β‐casein (Pohlmeier et al, 2011), bovine αs1‐casein (Ebert et al, 1994; Serova et al, 2012), and ovine β‐lactoglobulin (BLG; Barash et al, 1999; Reichenstein et al, 2005; Khodarovich et al, 2008), which contain binding sites for transcription factors such as the signal transducer and activator of transcription 5 (STAT5), CCAAT enhancer‐binding protein (C/EBP), nuclear factor I (NF‐I), glucocorticoid receptor (GR), E26‐specific sequence factor (Ets), and octamer‐binding transcription factor‐1 (Oct‐1; Reichenstein et al, 2005; Benlhabib and Herrera, 2006). Previous studies have shown that only a small percentage of cells in the mammary glands from a transgenic animal express recombinant protein, thus a more efficient mammary gland‐specific vector remains to be devised (Pantano et al, 2003; Zhou et al, 2012).…”

Section: Introductionmentioning

confidence: 99%

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary‐specific expression of human lactoferrin in transgenic mice

Cheng

Yuan

et al. 2012

Molecular Reproduction Devel

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It is very important to develop an effective, specific, and robust expression cassette that ensures a high level of expression in the mammary glands. In this study, we designed and constructed a series of mammary gland-specific vectors containing a complex hybrid promoter/enhancer by utilizing promoter sequences from milk proteins (i.e., goat β-casein, bovine αs1-casein, or goat β-lactoglobulin) and cytomegalovirus enhancer sequences; vectors containing a single milk protein promoter served as controls. Chicken β-globin insulator sequences were also included in some of these vectors. The expression of constructs was analyzed through the generation of transgenic mice. Enzyme-linked immunosorbent assay (ELISA) analysis revealed that the hybrid promoter/enhancer could drive the expression of recombinant human lactoferrin (rhLF) cDNA at high levels (1.17-8.10 mg/ml) in the milk of transgenic mice, whereas control promoters achieved a very low rhLF expression (7-40 ng/ml). Moreover, the expression of rhLF was not detected in the serum or saliva of any transgenic animal. This result shows that all constructs, driven by the hybrid promoter/enhancer, had high mammary gland-specific expression pattern. Together, our results suggest that the use of a hybrid promoter/enhancer is a valuable alternative approach for increasing mammary-specific expression of recombinant hLF in a transgenic mouse model.

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BLG‐e1 – A novel regulatory element in the distal region of the β‐lactoglobulin gene promoter

Cited by 4 publications

References 43 publications

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

Expression of Recombinant Human Alpha-Lactalbumin in the Milk of Transgenic Goats Using a Hybrid Pomoter/Enhancer

Tumors caused by overexpression and forced activation of Stat5 in mammary epithelial cells of transgenic mice are parity‐dependent and developed in aged, postestropausal females

Hybrid expression cassettes consisting of a milk protein promoter and a cytomegalovirus enhancer significantly increase mammary‐specific expression of human lactoferrin in transgenic mice

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