Name: in vitro bioluminescence assay to characterize circadian rhythm in mammary epithelial cells
Uploaded: 2017-09-28T12:30:00
Description: Watch this Scientific Journal Video about In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells at JoVE.com

이 작품으로는 2012 사회의 독성 (SOT) 지원 되었다-그렇지 연구 (M. 팡) Colgate Palmolive 그랜트와 국제 협력 연구 기금 동물 및 식물 검역 기관, 한국 공화국 (M. 팡), 그리고는 NIEHS 부여 P30ES005022 (H. Zarbl). 우리는 그의 도움이 토론, 씨 Shao-An 후안 그의 실험적인 지원을, 및 그녀의 증거 독서에 대 한 양 키 미 나카타 첸 청 (맥 거 번의과 대학 대학에서 휴스턴에서 건강 과학 센터 택 사스의) 감사 하 고 싶습니다.

1. PER2 발기인 구동 불안정 Luciferase 기자 벡터의 건설 <ol><li>pLS 사용자 정의 구입 [hPER2P / rLuc / 순수 하지 않아] 인코딩 rLuc 및 인간 cDNA를 포함 하는 벡터 PER2 발기인 조각 (hPER2P, 941 bp) Sac 사이 사이트에서 나 고 뒷 다리 여러 복제 지역 17에 III.</li>
	<li>인간의 PER2 발기인 조각 잘라 (hPER2P, 941 bp)는 벡터에서.
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포유류 세포 주기 circadian 클록의 상호 transcriptional/변환 피드백 루프에 의해 통제 된다. Heterodimers의 Bmal1 시계 및 Npas2 코어 CGs, Per2 , 외침및 수많은 CCGs4등의 발기인의 E 상자 요소에 바인딩하여 circadian 전사 조절. 그들은 셀, heterodimers의 축적으로 당: 외침 post-translationally 수정 및 시계: Bmal1 transcriptional 활동을 억 누르기 위해 핵 수송. 이 부정적인 피드백 루프 코어를 ...

Circadian 시계 넓은 범위 조절 유전자 약 24 h. 역학의 주기 예측 가능한 리듬 동작 자를 일주 식에서 생물 학적 과정의 연구 좋습니다 circadian 그 만성 장애 리듬의 유 방 및 교대 근로자, 간호사, 항공 승무원1,2,3등 전립선 암 위험을 증가 시킵니다. 이러한 결과 지속적인 빛, 밤에 빛, 또는 빛 사이클-시차 증가 종양 발생률을 모방 하 고 종양 성장을4,5가속을 노출을 보여주는 설치류 연구에 의해 뒷받침 됩니다. 인간과 설치류 학문에서 데이터를 바탕으로, 암 연구에 대 한 국제 기구의 분류는 가능성이 인간의 발암 물질 (유형 2A) 20106교대 작업.
우리는 설명 하는 이전에 유 방 종양 특정 발암 물질, Nnitroso의 단일 발암 복용량-N-methylurea (NMU) 중단 circadian 표현의 주요 circadian 유전자 (CGs) (예를 들어, 기간 2 , Per2) 여러 circadian 제어 유전자 (CCGs), 포함 한 DNA 손상 응답 키 및 대상 유선 (에 간) (DDRR) 유전자를 복구. 또한, 정상을 향해 Per2 와 DDRR 유전자의 circadian 식 식이 L의 chemopreventive 처방에 의해 다시 설정-메 틸-셀레노시스테인 (MSC) 63%로 종양의 발생률을 감소. 이러한 연구 결과 circadian 리듬, 화학 발암 및 자성7,8간의 기계적 링크를 표시 하는 첫번째 이었다. 기타 환경 유독 circadian 유전자 식에 vivo에서 방해를 표시 하는 노출 환경 질병9,10의 증가 위험과 관련 된 있습니다. 환경 유독 병 인 여 circadian 중단 연결 메커니즘을 이해 질병 예방에 mechanistically 기반 접근을 이어질 수 있습니다. 그러나, 연구는 노출 사이 상호 작용을 정의 목적 및 circadian 리듬은 일반적으로 수행 vivo에서. 24 또는 48 h 동안 모든 3-4 h를 수집는 전형적인 vivo에서 실험 조사 circadian 리듬에 미치는 영향 필요, 동물의 많은 수에서 3 개 이상 조직 제어 및 3 노출된 동물 되어야 합니다. Vivo에서 관찰 및 메커니즘이 검증 된 시험관에 시스템의 개발 따라서 뿐만 아니라, 동물의 수를 줄일 것 이라고 하지만 실험 비용 및 요구 사항 또한 극적으로 감소 하는 연구팀은 24-48 h 동안 지속적으로 작동합니다. 또한, 시스템 검증 된 시험관에 circadian 리듬, 또는 환경 스트레스 또는 유독 그것의 응답에 영향을 주는 유전 변경 화합물의 높은 처리량 검열 사용 될 수 있습니다. 따라서, 생체 외에서 그리고 vivo에서 모델과 실험의 협력 조합 다른 초점으로 다른 통찰력을 얻기 위해 필요 합니다.
포유류에서 circadian 발진기는 SCN의 특수 신경 뿐만 아니라 가장 주변 셀 형식에 존재 한다. 이 분자 시계는 비슷합니다 설립된 fibroblast 세포 선 및 기본 섬유 아 세포에서 배아 또는 성인 동물; 그러나, 조직 유형 특정 휴대 전화 모델11에 대 한 필요가 있다. 따라서, 운동 활동에서 vivo에서, SCN의 전통적인 연구 explants 비보 전, 그리고 불멸 하 게 fibroblast 세포의 체 외에서 세포 기반 분석은 널리 셀 자치 circadian 결함을 공부 하는 데 사용 됩니다. 그러나, 생체 외에서 fibroblast 세포 기반 분석 결과 circadian 메커니즘을 정리 수 있습니다 나타내는 증거가 없다 하 고 다른 주변 장기에 vivo에서의 셀에 응답 제시. 다른 세포 유형 유전자 발현, xenobiotic 물질 대사, 및 DDRR, 독특한 패턴을 가질 수 있으며 독성과 circadian 유전자 발현 사이의 링크 셀 형 수 있습니다 특정 및 다른 생리 적인 매개 변수에 의해 변조. 또한, 섬유 기반 시스템에서 circadian 발진기는 하지 되었습니다 완전히 평가 환경 유독, 스트레스 및 질병 개발 및 예방의 메커니즘에 노출 링크 예방 에이전트에 대 한 응답에 대 한. 따라서, 손쉬운, 유효한 셀 유형 전용, 생체 외에서 생물 발광 분석 실험 연구 기관 특정 환경 circadian 분리기에 대 한 필요가 있다. 다양 한 세포 시계 모델 (예를 들어, 간, keratinocytes, 지방 세포 뿐만 아니라 다리 셀 선)는 최근 몇 년 동안12,,1314, 에서 개발 되었습니다. 15, 여기서 설명 하는 분석 결과 유 방 상피 세포, 그리고 vivo에서 환경 스트레스, 유독, 마약과 chemopreventive 대리인 응답을 정리를 첫 번째 데모에서 첫 번째 세포 시계 모델입니다.
Renilla luciferase (rLuc) 및 반딧불 luciferase 30-61 kDa 단위체 단백질 효소 활동에 대 한 처리 posttranslational 필요 하지 않은 및 번역 즉시 유전 기자로 서 기능을 할 수 있습니다. 일단 기판 연결 luciferase 효소, 생 화 확 적인 반응을 촉매 빛의 플래시를 생성 합니다. 따라서, luciferase 구문은 진 식 기자 시스템에서 생체 외에서 그리고 에 비보로 널리 사용 됩니다. 그러나, circadian 리듬 연구, luciferase 기자의 유틸리티 luciferase 단백질의 상대적으로 긴 반감기에 의해 제한 됩니다 (T1/2 = 3.68 h) 기간 (특히 짧은 기간) circadian 유전자에 변화에 대 한 상대적인 식; 그러나, 수많은 연구 년 동안 성공적으로 사용 luciferase 유전자 빠르게 분해 luciferase 같은 circadian 리듬, 특히 오랜 기간 함께 리듬에 대 한 보고를 위해 필요 하지 않을 수 있음을 나타내는 pGL3 벡터에 24 h입니다. 따라서, 기자 플라스 미드 정한 luciferase 벡터를 사용 하 여,: pGL [Luc2P/Neo] hPEST (단백질 불안 시퀀스)를 포함 하는 개발 되었습니다, 현재 체 외 circadian 기자 벡터로 사용 하 수 있도록 생물 발광 분석 결과. Luc2P 에 의해 단백질은 훨씬 더 짧은 반감기 (T1/2 = 0.84 h) 따라서, 응답 보다 신속 하 고 더 큰 크기와 변화에 야생-타입 보다 transcriptional 활동에 나그것은 될 수 있는 ndicating luciferase 실시간16에 정확 하 게 PER2 발기인에 의해 규제의 리듬 표현 모니터링 하는 데 사용.

<table>
	<tbody>
		<tr>
			<td>pLS[hPER2P/rLuc/Puro] vector</td>
			<td>SwitchGear Genomics</td>
			<td>S700000</td>
			<td>customized vector</td>
		</tr>
		<tr>
			<td>pGL4.18[Luc2P/Neo] vector</td>
			<td>Promega</td>
			<td>9PIE673</td>
			<td>destabilized luciferase expression vector</td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>Invitrogen</td>
			<td>15224041</td>
			<td>For subcloning</td>
		</tr>
		<tr>
			<td>TOPO TA cloning kit</td>
			<td>Invitrogen</td>
			<td>K4500-02</td>
			<td>with One Shot TOP10 Chemically Competent E. coli</td>
		</tr>
		<tr>
			<td>Sac I</td>
			<td>New England BioLabs</td>
			<td>R0156S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>Hind III-HF</td>
			<td>New England BioLabs</td>
			<td>R3104S</td>
			<td>Restriction enzyme</td>
		</tr>
		<tr>
			<td>CutSmart buffer</td>
			<td>New England BioLabs</td>
			<td>B7204S</td>
			<td>Restriction enzyme buffer</td>
		</tr>
		<tr>
			<td>DNA gel extraction kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td>Purify DNA fragments from agarose gel</td>
		</tr>
		<tr>
			<td>PCR Purification Kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>DNA clean up</td>
		</tr>
		<tr>
			<td>LB Miller&#39;s modification</td>
			<td>TEKNOVA</td>
			<td>L8600</td>
			<td>For transformed E. Coli culture</td>
		</tr>
		<tr>
			<td>LB Agar Plate</td>
			<td>TEKNOVA</td>
			<td>L1902</td>
			<td>For white/blue selection</td>
		</tr>
		<tr>
			<td>QIAprep spin miniprep Kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td>For extraction of plasmide DNA</td>
		</tr>
		<tr>
			<td>EndoFree plasmid maxi prep Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>For extraction of plasmide DNA</td>
		</tr>
		<tr>
			<td>FuGene HD</td>
			<td>Promega</td>
			<td>E2311</td>
			<td>Transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM reduced serum medium</td>
			<td>Invitrogen</td>
			<td>31985-062</td>
			<td>For transfection</td>
		</tr>
		<tr>
			<td>MCF10A cell line</td>
			<td>American Type Culture Collection</td>
			<td>CRL-10317</td>
			<td>Mammary Epithelial Cells</td>
		</tr>
		<tr>
			<td>MEGM BulletKit</td>
			<td>Lonza</td>
			<td>CC-3150</td>
			<td>Mammary Epithelial Growth Medium</td>
		</tr>
		<tr>
			<td>MEBM</td>
			<td>Lonza</td>
			<td>CC-3151</td>
			<td>Mammary Epithelial Basal Medium</td>
		</tr>
		<tr>
			<td>MEGM SingleQuot Kit</td>
			<td>Lonza</td>
			<td>CC-4136</td>
			<td>Suppliments &#38; Growth Factors</td>
		</tr>
		<tr>
			<td>ReagentPack</td>
			<td>Lonza</td>
			<td>CC-5034</td>
			<td>Reagent for subculture</td>
		</tr>
		<tr>
			<td>D-PBS (10X)</td>
			<td>Sigma</td>
			<td>D1408</td>
			<td>Wash cells in culture dishes</td>
		</tr>
		<tr>
			<td>UltraPure Distilled Water</td>
			<td>Invitrogen</td>
			<td>10977</td>
			<td>Dilute 10X D-PBS</td>
		</tr>
		<tr>
			<td>Tissue culture dish (35 mm)</td>
			<td>BD/Falcon</td>
			<td>353001</td>
			<td>cell culture dish suitable to LumiCycle</td>
		</tr>
		<tr>
			<td>Silicon grease</td>
			<td>Fisher</td>
			<td>NC9044707</td>
			<td>For sealing dish with recording medium</td>
		</tr>
		<tr>
			<td>Round cover glass</td>
			<td>Harvard Bioscience</td>
			<td>64-1500 (CS-40R)</td>
			<td>For sealing dish with recording medium</td>
		</tr>
		<tr>
			<td>Cholera toxin</td>
			<td>Sigma</td>
			<td>C8052</td>
			<td>Supplement for growth medium</td>
		</tr>
		<tr>
			<td>G418 sulfate (Geniticin)</td>
			<td>Invitrogen</td>
			<td>10131035</td>
			<td>Antibiotic for selection of stabliy transfected cells</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A9393</td>
			<td>For colony selection</td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma</td>
			<td>F6886</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Melatonin</td>
			<td>Sigma</td>
			<td>M5250</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma</td>
			<td>H1138</td>
			<td>Synchronization agent</td>
		</tr>
		<tr>
			<td>d-Luciferin, sodium salt</td>
			<td>Invitrogen</td>
			<td>L2912</td>
			<td>Luciferase substrate</td>
		</tr>
		<tr>
			<td>IC261</td>
			<td>Sigma</td>
			<td>10658</td>
			<td>Positive control for circadian disruptor</td>
		</tr>
		<tr>
			<td>Methylnitrosourea (NMU)</td>
			<td>Sigma</td>
			<td>N1517</td>
			<td>Mammary specific carcinogen</td>
		</tr>
		<tr>
			<td>Methylselenocysteine (MSC)</td>
			<td>Sigma</td>
			<td>M6680</td>
			<td>Organic selenium (chemopreventive agent)</td>
		</tr>
		<tr>
			<td>EX527</td>
			<td>Sigma</td>
			<td>E7034</td>
			<td>SIRT1 specific inhibitor</td>
		</tr>
		<tr>
			<td>Cambinol</td>
			<td>Sigma</td>
			<td>C0494</td>
			<td>SIRT1 &#38; SIRT2 inhibitor</td>
		</tr>
		<tr>
			<td>NanoDrop Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop 8000</td>
			<td>Quantify nucleotide</td>
		</tr>
		<tr>
			<td>GeneAmp PCR System 9700</td>
			<td>Applied Biosystems</td>
			<td>N805-0200</td>
			<td>For molecular biology experiment</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NAPCO</td>
			<td>Series 8000 DH</td>
			<td>For cell culture at 5% CO2 at 37 &#176;C</td>
		</tr>
		<tr>
			<td>Desktop centrifuge with refrezerator</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td>For molecular biology experiment</td>
		</tr>
		<tr>
			<td>Centrifuge with swing bucket</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>80124</td>
			<td>Phase contrast optional</td>
		</tr>
		<tr>
			<td>Tissue culture hood</td>
			<td>Labconco</td>
			<td>Class II A2</td>
			<td>BSL-2 certified</td>
		</tr>
		<tr>
			<td>LumiCycle 32</td>
			<td>Actimetrics</td>
			<td>Not Available</td>
			<td>Luminoscence detector</td>
		</tr>
	</tbody>
</table>

in vitro bioluminescence assay to characterize circadian rhythm in mammary epithelial cells

Circadian 리듬 동작 자 유전자 발현에서 배열 하는 생물 학적 과정을 조절 하는 기본적인 생리 과정 모든 생물에 존재 이다. 척추 동물, circadian 리듬 (SCN, 중앙 맥 박 조정기) suprachiasmatic 핵 및 가장 주변 조직을 구성 하는 개별 셀 기능 분자 발진기에 의해 제어 됩니다. 더 중요 한 것은, 밤에 빛, 환경 스트레스 또는 유독 하 여 circadian 리듬의 만성 질환과 노화의 위험 증가와 연결 됩니다. 중앙 및 주변 생물 학적 시계를 중단 시킬 수 있는 에이전트와 에이전트 방지 또는 circadian 중단의 효과 완화 수 있는 식별 수 만성 질환의 예방에 중요 한 의미를 갖는다. 설치류 모델 노출 및 유도 또는 circadian 중단 방지/완화 하는 에이전트를 식별 하는 데 수 있습니다, 비록이 실험 동물의 많은 수를 필요 합니다. Vivo에서 연구는 또한 중요 한 자원과 인프라를 요구 하 고 밤새 일 하는 연구원을 필요로. 따라서, 있다 시스템 적절 한 체 외에서 세포 형에 대 한 긴급 한 필요 환경 circadian 분리기 및 강화 셀 형식에 대 한 화면을 다른 기관과 질병 상태에서. 우리는 인간 기간 2 유전자 발기인의 제어에서 진 핵 세포에서 정한 luciferase의 전사를 구동 하는 벡터를 건설 한다. 이 circadian 기자 구조는 안정적으로 인간 유 방 상피 세포로 페 그리고 circadian 응답 기자 세포 생체 외에서 생물 발광 분석 결과 개발 하기 위해 선정 됐다. 여기, 우리는 상세한 프로토콜을 설정 하 고 분석 결과 제시. 우리는 더는 vivo에서 다양 한 화학 물질의 효과 세포 생물 학적 시계에 정리 분석 결과 생체 외에서 우리의 수를 보여주는 개념 실험의 증거에 대 한 세부 정보를 제공 합니다. 결과 분석 결과 다양 한 종류의 세포에 대해 환경 분리기 및 circadian clock의 chemopreventive 강화를 적용할 수 있습니다 나타냅니다.

Circadian 생물 발광 기자 벡터: 인간의 PER2 발기인 기반 표현의 정한 luciferase 변종
941 bp 조각으로 구성 된 DNA 순서: pGL circadian 기자 벡터를 생성 하는 데 사용 하는 인간의 PER2 발기인에서 파생 된 [hPer2P/Luc2P/Neo, 처음 규제 요소 규제 알려진 존재에 대 한 분석 circadian 진 식입니다. 생물 정보학 분석이 보여?...

Watch this Scientific Journal Video about In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells at JoVE.com

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

생체 외에서 생물 발광 분석 결과 유 방 상피 세포에서 세포 circadian 리듬을 결정 하기 위해 제공 됩니다. 이 메서드는 기간 2 유전자 발기인의 제어에서 정한 luciferase 표현 하는 포유류 세포 기자 플라스 미드를 활용 합니다. 그것은 다른 세포 유형 circadian 리듬에 기관 특정 효과 평가 하 게 적응 될 수 있다.

생체 외에서 유 방 상피 세포에서 Circadian 리듬 특성 분석 결과 생물 발광

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

The overall goal of this in vitro bioluminescence assay is to determine the effects of different environmental circadian disruptors and enhancers on the cellular circadian rhythm of mammary epithelial cells from mammary glands at normal or diseased states. This method can help answer key questions in the environmental health field such as whether environmental factors including chemical compounds affect the cellular circadian rhythm. So, the main advantage of this assay is that it can be readily adapted to a variety of cell types so we can screen for environmental modulators of circadian clocks in different organs.Demonstrating the procedure with me is Brian Estrella, a graduate student in our lab. The human PER2 promoter fragment is obtained from a customized vector that contains human PER2P at the site between SecI and HindIII in the multiple cloning region. To cut the human PER2P fragment out from the vector, mix the following in a DNA/RNA and nucleotide-free microcentrifuge tube, 2.5 microliters of restriction enzyme buffer, 10 microliters of vector, one microliter of SacI restriction enzyme, one microliter of HindIII restriction enzyme, and ultrapure water to 25 microliters.Incubate in a heating block at 37 degrees Celsius for 90 minutes. When the restriction digest is complete, run the whole volume of the reaction on a 0.7%agarose gel containing 0001%ethidium bromide to separate human PER2P from the vector. Cut a piece of agarose gel containing a fluorescence band at 941 base pairs, purify the human PER2P fragments from the gel, and quantify the DNA as described in the text protocol.The next step is to clone human PER2P into a vector with destabilized fire flow luciferase. Linearize one microgram of the vector by restriction digest with SacI and HindIII as shown previously. Run the whole volume of the reaction on a gel containing ethidium bromide and then purify and quantify the vector DNA as before.To ligate human PER2P into the linearized vector, gently mix the following in a microcentrifuge tube, two microliters of T4 DNA ligase reaction buffer, 3.26 microliters of human PER2P, one microliter of linearized vector, and water to 20 microliters. Then add one microliter of T4 DNA ligase, mix gently, and incubate at 16 degrees Celsius in a thermal cycler overnight. On the following day, chill the ligated vector on ice.Prior to the transformation, set a water bath at 42 degrees Celsius, warm up SOC medium at room temperature, and warm up LB agar plates in the 37 degree Celsius incubator. Thaw two vials of competent E.coli cells on ice. To start the transformation, add six microliters of ligated vector to one tube of chemically competent E.coli and gently flip the tube to mix.As a negative control, add one microliter of empty vector to another tube of chemically competent E.coli and incubate the tubes on ice for 30 minutes. After 30 minutes, heat shock in the 42 degrees Celsius water bath for 30 seconds without shaking and then return the tubes to ice. Add 250 microliters of SOC medium to each tube and incubate at 37 degrees Celsius with shaking at 200 rpm for one hour.Next, evenly spread 10 to 50 microliters of each transformed E.coli reaction on the surface of an LB agar plate. Put the plates upside down in a 37 degree Celsius incubator overnight. On the following day, use a sterilized inoculating loop to pick three to six white colonies from each plate and release each colony into a culture tube containing four milliliters of LB culture medium with 50 micrograms per milliliter of ampicillin.Incubate the tubes at 37 degrees Celsius with shaking at 200 rpm overnight. Use two milliliters of each four milliliters of cultured E.coli for DNA extraction. After verifying the insert as described in the text protocol, amplify the selected E.coli by adding the leftover two milliliters of cultured E.coli into 200 milliliters of LB culture medium containing 50 micrograms per milliliter of ampicillin and incubating at 37 degrees Celsius with shaking at 200 rpm overnight.Plasma DNA is then extracted with an endotoxin-free plasmid Maxiprep kit per the manufacturer's instruction. After quantifying the plasma DNA, aliquot and store at minus 80 degrees Celsius for future use. The mammary epithelial cells to be transfected with the constructed circadian vector are cultured with mammary epithelial cell growth medium containing mammary epithelial basal medium, growth supplements, and cholera toxin.To dissociate the cells, incubate with 10 milliliters of Trypsin-EDTA for about 20 minutes and then neutralize the Trypsin by adding 10 milliliters of Trypsin neutralizing solution. Transfer the cells to a 50 milliliter centrifuge tube. Rinse the dish with 10 milliliters of buffered saline solution and add the rinse to the 50 milliliter tube.Spin down the cells at 1, 500 rpm for 10 minutes. Remove the supernatant and add three milliliters of MEGM to resuspend the cells. Count the cells with a hemacytometer under an inverted microscope.After calculating the concentration of the single cell suspension, seed two times 10 to the fifth cells evenly per 35 millimeter culture dish with MEGM. Swirl the plates thoroughly to obtain an even distribution of cells in each plate. Grow the cells in the cell culture incubator to 20 to 30%confluence.On the following day, warm up reduced serum medium in a 37 degree Celsius water bath and the transfection reagent at room temperature for 30 minutes. Bring the plasma DNA to room temperature. Dilute the plasma DNA to 30 nanograms per microliter with endotoxin-free Tris-EDTA buffer and keep at room temperature.Prepare each plasmid transfection mixture in a 1.5 milliliter microcentrifuge tube. First, add 63.6 microliters of warm reduced serum media. Then, add 33.4%microliters of plasma DNA and mix gently by pipetting.Lastly, add three microliters of transfection reagent directly into the center of the tube without touching the wall. Mix gently by pipetting and keep at room temperature for 30 minutes. Drop the entire plasma transfection mixture directly onto the center surface of the medium in the plate and mix gently by shaking the dish.Culture the cells in a carbon dioxide incubator for an additional 48 to 72 hours. 48 to 72 hours after transfection when the cultured cells are about 70%confluent, replace the medium with MEBM without growth factors and culture the cells for an additional 24 hours. After 24 hours of starvation, add a synchronization agent in MEBM to the cells.50%horse serum is used as the synchronization agent in this experiment. Incubate for 1-1/2 hours in a carbon dioxide incubator. Prepare recording medium.When the treatment with the synchronization agent is complete, wash the cells three times with warm D-PBS. Then add two milliliters of freshly prepared recording medium containing luciferin. Seal the dish with a sterilized cover glass using silicone grease to prevent evaporation.Label the dish on the side. Place the sealed dish in the seat inside the luminometer and perform data collection and analysis as described in the text protocol. In this in vitro model, untreated transiently transfected cells showed at least two complete cycles of luminescence signaling after synchronization.Cells exposed to the mutagen NMU showed disrupted circadian rhythm at 0.5 millimolar concentration as reflected by the loss of luminescence signaling after synchronization. Inhibition of Cert1 activity with X257 or Cambinol similarly disrupted circadian rhythm by dampening the subsequent circadian cycle. Importantly, the addition of chemopreventive MSC to the culture medium restored the rhythm disrupted by 0.5 millimolar NMU and prevented the disruptive effects of the Cert1 inhibitors.In stably transfected cells, both NMU and Cert1 inhibitors disrupted circadian rhythm. MSC restored circadian rhythms in cells pretreated with NMU, X275, or Cambinol. The bioluminescence intensity was higher but the rhythm was sustained for a shorter time in transiently transfected versus stably transfected cells, but the overall results are consistent.The cellular circadian rhythm can be quantified in circadian parameters such as period and phase using LumiCycle analysis software. Once mastered, this technique is simple, fast, and safe. While attempting this procedure, it's important to track if the cells are proliferating since the transfection efficiency is very low in nondividing cells such as neuronal cells in which case a virus-spaced transfection method could be considered.After transfection, other methods like treatment with different chemicals or genetic modification can be performed to answer additional questions like how is the cellular circadian rhythm affected by environmental and genetic factors. This assay can be used to investigate the impact of disrupted circadian rhythm on cellular function. By studying the underlying mechanisms, we can develop better intervention strategies for those at increased risk of breast cancer due to abnormal work schedules.After watching this video, you should have a good understanding of how to establish and validate the in vitro bioluminescence assay to determine the circadian rhythm in the cells of interest. Don't forget that working with a UV illuminator can be extremely hazardous and precautions such as using a UV protective face shield should always be taken while performing this procedure. Thanks for watching and good luck with your experiments.

Research

JoVE Journal

Genetics

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

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