Cloning and Molecular Characterization of Porcine β-casein Gene (CNS2)

Lee, Sang-Mi,Kim, Hye-Min,Moon, Seung-Ju,Kang, Man-Jong Asian Australasian Association of Animal Productio 2012 Animal Bioscience Vol.25 No.3

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Milk is presently the most mature system for production of therapeutic proteins from a transgenic animal. Specifically, ${\beta}$-casein is a major component of cow, goat and sheep milk, and its promoter has been used to regulate the expression of transgenic genes in the mammary gland of transgenic animals. Here, we cloned the porcine ${\beta}$-casein gene and analyzed the transcriptional activity of the promoter and intron 1 region of the porcine ${\beta}$-casein gene. Sequence inspection of the 5'-flanking region revealed potential DNA elements including SRY, CdxA, AML-a, GATA-3, GATA-1 and C/EBP ${\beta}$. In addition, the first intron of the porcine ${\beta}$-casein gene contained the transcriptional enhancers Oct-1, SRY, YY1, C/EBP ${\beta}$, and AP-1, as well as the retroviral TATA box. We estimated the transcriptional activity for the 5'-proximal region with or without intron 1 of the porcine ${\beta}$-casein gene in HC11 cells stimulated with lactogenic hormones. High transcriptional activity was obtained for the 5'-proximal region with intron 1 of the porcine ${\beta}$-casein gene. The ${\beta}$-casein gene containing the mutant TATA box (CATAAAA) was also cloned from another individual pig. Promoter activity of the luciferase vector containing the mutant TATA box was weaker than the same vector containing the normal TATA box. Taken together, these findings suggest that the transcription of porcine ${\beta}$-casein gene is regulated by lactogenic hormone via intron 1 and promoter containing a mutant TATA box (CATAAAA) has poor porcine ${\beta}$-casein gene activity.

Mouse Mammary Epithelial Cell에서 Retrovirus Vector를 이용한 Human Lactadherin 유전자의 유도적 발현

권모선,구본철,정병현,염행철,박창식,김태완 한국동물생명공학회(구 한국동물번식학회) 2003 Reproductive & developmental biology Vol.27 No.1

본 연구는 VSV-G glycoprotein을 envelope으로 하는 pseudotyped retrovirus vector system을 이용하여 쥐의 유방상피세포인 HC11에서 human Lactadherin 유전자의 발현을 확인하고자 하였다. 실험에 사용한 vector는 개체내에서의 외래 유전자의 지속적인 발현에 의한 생리적인 부작용을 최소화하기 위한 구조로, 조직특이적이며 lactogenic hormone에 의해 유도적인 활성을 가지는 것으로 알려진 WAP promoter의 통제하에 도입하고자 하는 외래 유전자를 위치하도록 하였다. WAP promoter의 대조군으로 지속적인 활성을 나타내는 β-actin promoter를 사용하였으며, 이 각각의 promoter와 marker gene으로 E. coli LacZ gene을 재조합한 후 retrovirus vector system을 이용하여 HCll에 도입하였다. 세포의 genome 내로의 유전자의 전이는 PCR을 통해 확인하였고, RT-PCR의 수행으로 유전자의 발현을 확인하였다. Lactadherin 유전자를 이용한 실험도 동일한 과정으로 수행하였으며, RT-PCR의 결과에서 HCll 세포에서 Lactadherin 유전자의 발현이 insulin을 단독으로 처리한 군에 비해 insulin, hydrocortisone, prolactin을 동시에 처리한 군에서 우월하게 나타나는 것으로 확인되었다. 그러나 insulin 단독 처리군에서 유전자의 발현이 약하게 나타나는 것으로 관찰되어 WAP promoter의 leakiness에 대한 재고의 필요성이 요구되었다. Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinomas and may prevent symptomatic rotavirus infections. In this study, under the control of tissue specific and hormonal inducible mouse whey acidic protein (WAP) promote., the expression pattern of lactadherin (Ltd) in lactogenic hormone-dependent mouse mammary epithelial cell line HC11 were tested. pLNWLtd construct containing 2.4 kilobases of the WAP promoter and 1.5 kilobases of human lactadherin gene was stably transfered into HC11 cells using retroviral vector system. Integration and expression level of the transgene was estimated using PCR and RT-PCR, respectively. Prominent induction of Ltd gene under the WAP promoter was accomplished in the presence of insulin, hydrocortisone and prolactin. Compared to the control (cells cultured with insulin alone), however we observed that the WAP promoter was leaky. These data indicate that luther studies are needed in finding an appropriate promoter other than WAP promoter because of its leakiness.

염소의 베타-락토글로불린 유전자 프로모터 활성의 호르몬에 의한 조절

김재만,김경진 한국통합생물학회 1995 동물학회지 Vol.38 No.3

유선 조직에서 베타-락토글로불린 유전자의 발현은 프롤락틴, 글루코코르티코이드 및 인슐린등의 유촉진 호르몬들에 의해서 강력하게 유도된다. 이와 같은 호르몬 유도의 조절 기작을 규명하기 위하여, 배양 유선 세포인 HC11 세포에서 염소 베타-락토글로불린 유전자 프로모터의 유촉진 호르몬에 대한 반응을 분석하였다. 베타-락토글로불린 프로모터의 5'- 조절 부위를 연쇄적으로 제거한 발현 실험에서 호르몬 유도를 크게 변화 시키는 두 지역이 관찰되었다. 조절 부위의 -1692의 상류지역은 하류 프로모터를 강력하게 활성화 시키는 부위로, 주로 글루코코르티코이드 유도체인 덱사메타손의 작용을 매개하였다. 그러나 두번째 지역의 유도 작용은 인슐린 처리를 병행하지 않을 경우 상류 조절부위에 의해 억제되었다. 이러한 결과는, 유선세포에서 유촉진 호르몬들에 의한 베타-락토글로불린 프로모터 활성 유도가 인슐린에 의한 탈 억제화와 글루코코르티코이드 및 프롤락틴에 의한 활성화의 복합 조절에 의해서 이루어질 것이라는 점을 시사한다. 두번째 지역에 의한 덱사메타손 유도는 -700 부근의 글루코코르티코이드 수용체 결합 부위에 의해서 매개되는 것으로 추정된다. Expression of $\beta$-lactoglobulin gene in mammary tissue is strongly induced by lactogenic hormones such as prolactin, glucocorticoid, and insulin. In order to elucidate the regulatory mechanism underlying such hormonal induction, the response of the caprine $\beta$-lactoglobulin gene promoter to lactogenic hormones was analyzed in cultured HC11 mammary cells. Expression with serial deletions of the 5' -regulatory sequence of the $\beta$-lactoglobulin promoter revealed that two regions are responsible for a substantial change in hormonal indudbility. The region upstream of-1692, which exhibited strong repression of the downstream promoter, mediated the induction by insulin. This insulin-response was independent of the other two lactogenic hormones, prolactin and glucocorticoid. The other region from -740 to -470, which showed strong activation of the $\beta$-lactoglobulin promoter in confluent HC11 mammary cells, mediated mainly the response to a glucocorticoid analogue, dexametasone. The induction by the latter region, however, was suppressed by the usptream repression without insulin treatment. These results suggest that the induction of $\beta$-lactoglobulin promoter activity by lactogenic hormones in mammary cells may be achieved by the combined action of derepression by in sulin and activation by glucocorticoid and prolactin. Dexametasone response by the latter region seems to be mediated by the glucocorticoid receptor site around -7OObp.

Lactogenic Activity of an Enzymatic Hydrolysate from Octopus vulgaris and Carica papaya in SD Rats

Bingna Cai,Hua Chen,Han Sun,Huili Sun,Peng Wang,Deke Chen,Jianyu Pan 한국식품영양과학회 2015 Journal of medicinal food Vol.18 No.11

The traditional Chinese medicine theory believes that octopus papaya soup can stimulate milk production in lactating women. The objective of this study was to determine whether dietary supplementation with an enzymatic hydrolysate of Octopus vulgaris and Carica papaya (EHOC) could increase milk production and nutritional indexes in Sprague Dawley (SD) rats. Female SD rats (n = 24) were fed a control diet (n = 8), EHOC-supplemented diet, or a positive control diet (Shengruzhi) from day 10 of pregnancy to day 10 of lactation. Maternal serum, mammary gland (day 10 of lactation), milk, and pup weight (daily) were collected for analysis. Results showed that the EHOC diet obviously elevated daily milk yield and pup weight compared to the control group (P < .05). The EHOC diet was found to increase the concentration of prolactin (PRL), progesterone (P), estradiol (E2), and growth hormone (GH) significantly in the circulation and mammary gland. Mammary glands of EHOCtreated dams showed clear lobuloalveolar development and proliferation of myoepithelial cells, but no striking variations were observed among the groups. Furthermore, the nutrition content and immune globulin concentration in the milk of EHOCsupplemented dams were higher than those of the control group, especially the cholesterol, glucose, and IgG were higher by 44.98% (P < .001), 42.76% (P< .01), and 42.23% (P < .01), respectively. In conclusion, this article demonstrates that EHOC administration has beneficial effects on milk production in the dams and on performance of the dam and pup. These results indicate that EHOC could be explored as a potentially lactogenic nutriment for lactating women.

Gene expression and hormonal regulation of adiponectin and its receptors in bovine mammary gland and mammary epithelial cells

OHTANI, Yoshihisa,YONEZAWA, Tomo,SONG, Sang‐,Houn,TAKAHASHI, Tatsuyuki,ARDIYANTI, Astrid,SATO, Katsuyoshi,HAGINO, Akihiko,ROH, Sang‐,gun,KATOH, Kazuo Blackwell Publishing Asia 2011 Animal science journal Vol.82 No.1

<P><B>ABSTRACT</B></P><P>Although the functions of adiponectin, a differentiated adipocyte‐derived hormone, in regulating glucose and fatty acid metabolism are regulated by two subtypes of adiponectin receptors (AdipoRs; AdipoR1 and AdipoR2), those in ruminants remain unclear. Therefore we examined the messenger RNA (mRNA) expression levels of adiponectin and its receptors in various bovine tissues and mammary glands among different lactation stages, and the effects of lactogenic hormones (insulin, dexamethasone and prolactin) and growth hormone (GH) on mRNA expression of the AdipoRs in cultured bovine mammary epithelial cells (BMEC). AdipoRs mRNAs were widely expressed in various bovine tissues, but adiponectin mRNA expression was significantly higher in adipose tissue than in other tissues. In the mammary gland, although adiponectin mRNA expression was significantly decreased at lactation, AdipoR1 mRNA expression was significantly higher at peak lactation than at the dry‐off stage. In BMEC, lactogenic hormones and GH upregulated AdipoR2 mRNA expression but did not change that of AdipoR1. In conclusion, adiponectin and its receptor mRNA were expressed in various bovine tissues and the adiponectin mRNA level was decreased during lactation. These results suggest that adiponectin and its receptors ware changed in mammary glands by lactation and that AdipoRs mRNA expression was regulated by different pathways in BMEC.</P>

Mechanism and Regulation of Amino Acid Transport in Mammary Gland - Review -

Kansal, Vinod K.,Sharma, Rekha Asian Australasian Association of Animal Productio 2001 Animal Bioscience Vol.14 No.5

Several amino acid transport systems in mammary gland have been characterized during the last few years. These systems may be divided into two broad categories based on whether they are sodium-dependent or $Na^{+}$-independent, and each of these categories is subdivided into 3 groups depending on whether the systems prefer zwitterionic, cationic or anionic substrates. The zwitterion preferring transport processes in mammary gland are $Na^{+}$-dependent system A and $Na^{+}$-independent systems L and T. System $y^{+}$ is a $Na^{+}$-independent transporter of cationic amino acids and $X_{AG^{-}}$ is a $Na^{+}$-dependent system for anionic amino acids. A ($Na^{+}+Cl^{-}$)-dependent system, selective for $\beta$-amino acids has been reported in rat mammary tissue. In addition, there is yet another class of transporters that have still broader specificity. The $Na^{+}$-dependent systems $BCl^{-}$-dependent and $BCl^{-}$-independent and $Na^{+}$-independent system $y^{+}L$ have been reported to mediate the transport of zwitterionic as well as cationic amino acids. Each system has been characterized with respect to its substrate specificity, affinity, kinetics and ion-dependence. Transport of amino acids by mammary tissue is regulated by i) the intracellular substrate concentration, ii) lactogenic hormones and iii) milk stasis. Four of the above transport systems (i.e. A, L, $y^{+}$ and $BCl^{-}$-independent) are up-regulated by lactogenic hormones (insulin, cortisol and prolactin) in mammary gland.

Mouse Mammary Epithelial Cell에서 Retrovirus Vector를 이용한 Human Lactadherin 유전자의 유도적 발현

권모선,구본철,정병현,염행철,박창식,김태완 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinomas and may prevent symptomatic rotavirus infections. In this study, under the control of tissue specific and hormonal inducible mouse whey acidic protein (WAP) promoter, the expression pattern of lactadherin (Ltd) in lactogenic hormone - dependent mouse mammary epithelial cell line HCll were tested. pLNWLtd construct containing 2.4 kilobases of the WAP promoter and 1.5 kilobases of human lactadherin gene was stably transfered into HCll cells using retroviral vector system. Integration and expression level of the transgene was estimated using PCR and RT-PCR, respectively. Prominent induction of Ltd gene under the WAP promoter was accomplished in the presence of insulin, hydrocortisone and prolactin. Compared to the control (cells cultured with insulin alone), however we observed that the WAP promoter was leaky. These data indicate that futher studies are needed in finding an appropriate promoter other than WAP promoter because of its leakiness.

ACTINOMYCIN D AND LACTOGENIC HORMONES MODULATE CASEIN GENE EXPRESSION IN BOVINE ACINAR CULTURE

Choi, Y.J.,Woo, J.H.,Park, C.S.,Kim, Y.K. Asian Australasian Association of Animal Productio 1989 Animal Bioscience Vol.2 No.3

Inductional Expression of the Human Lactadherin Gene in Mouse Mammary Epithelial Cells

Kwon, Mo Sun,Koo, Bon Chul,Kim, Teoan 충남대학교 형질전환복제돼지연구센터 2004 논문집 Vol. No.8