Name: massively parallel reporter assays in cultured mammalian cells
Uploaded: 2014-08-17T16:00:00.000+00:00
Duration: 11 min 3 s
Description: Watch this Scientific Journal Video about Massively Parallel Reporter Assays in Cultured Mammalian Cells at JoVE.com

이 작품은 상을 수 R01HG006785에서 국립 보건원 (NIH)의 국립 인간 게놈 연구소에 의해 지원되었다.

1 시퀀스 설계 및 합성 <ol><li> 시퀀스의 설계 및 합성에 MPRA 시작은 규제 활동을 분석합니다. pMPRA 벡터 시리즈와의 호환성을 위해 다음과 같은 템플릿을 사용하여 각 시퀀스를 설계 : 5&#39;-ACTGGCCGCTTCACTG- var에 -GGTACCTCTAGA- 태그 -AGATCGGAAGAGCGTCG-3 var에 정량 할 수있는 순서를 나타내며, 태그는 하나 이상의 식별 태그를 의미한다 &#39;. </li><li> 이 가변 영역 (VAR 및 태그) 사이 리포터 유전자 단편의 방향을 용이하게 결찰를 KpnI (GGTACC)과를 XbaI (TCTAGA) 제한 부위의 쌍에 의해 분리된다. 또한, 두 가지 SfiI (GGCCNNNNNGGCC) 사이트는 pMPRA 백본으로 방향 결찰 할 수 있도록 PCR에 의해 추가됩니다. 변수 영역이 제한 부위의 사본을 추가로 포함 할 수 없습니다. 필요한 경우, 이들 제한 효소 중 하나 이상을 대체 할 수있다만큼 대체품으로서 1) 주변 DNA 분자의 말단에 효율적인 절단을 허용하고, 2) 벡터 시퀀스의 다른 곳에서 절단되지 않는다. 자세한 내용에 대한 설명을 참조하십시오. </li><li> 상용 공급 업체에서 표 1에 필요한 프라이머를 얻습니다. </li></ol><table border="0" cellpadding="0" cellspacing="0" width="381"><tbody><tr><td> MPRA_SfiI_F </td><td> GCTAAGGGCCTAACTGGCCGCTTCACTG </td></tr><tr><td> MPRA_SfiI_R </td><td> GTTTAAGGCCTCCGTGGCCGACGCTCTTC </td></tr><tr><td> TAGseq_P1 </td><td> AATGATACGGCGACCACCGAGATCTACACT CTTTCCCTACACGACGCTCTTCCGATCT </td></tr><tr><td> TAGseq_P2 </td><td> CAAGCAGAAGACGGCATACGAGAT [인덱스] GT GACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGAGG </td></tr></tbody></table> 표 1 프라이머 서열. [색인] 다중화 시퀀싱에 사용될 6-8 NT 인덱스 시퀀스를 나타낸다. 다른 인덱스 적어도 8 TagSeq-P2 프라이머를 얻습니다. 모든프라이머의 HPLC 또는 PAGE로 정제해야한다. <ol start="4"><li> 올리고 뉴클레오티드 라이브러리는 상업적 공급 업체 또는 코어 설비로부터 얻어진, 또는 제조자에 의해 설명 된 바와 같이 프로그램 가능한 마이크로 어레이 계 올리고 뉴클레오티드 합성기를 사용하여 생성 될 수있다. 100 ㎕의 TE 0.1 버퍼에 OLS 제품을 재현 탁. </li><li> 폴리 아크릴 아미드 겔을 변성 10 % TBE-우레아에 올리고 뉴클레오티드 라이브러리를 실행. 품질 (그림 2A)를 평가하는 ssDNA의 민감한 형광 얼룩 차선 얼룩 당 약 1 pmol의를 넣습니다. </li><li> 전체 길이의 올리고 뉴클레오티드에 대응 이산 밴드 시각화 할 수있는 경우 (도 2a, 레인 1과 2), 대응 아크릴 아미드 겔 슬라이스를 절제하고 진탕하면서 실온에서 밤새 100 ㎕의 TE 0.1로 올리고 뉴클레오티드를 용출. 그렇지 않으면, 원시 OLS 서스펜션을 사용하여 진행합니다. </li><li> PCR 증폭 9 에멀젼을 이용한 올리고 뉴클레오티드 라이브러리 SfiI 꼬리를 추가 </<html> &gt; SUP. </li><li> 표 2에 설명 된대로 50 ㎕의 PCR 반응 혼합물을 설정합니다. 다음 4 ℃에서 5 분 동안 300 ㎕의 Micellula DNA 에멀젼에서 오일과 계면 활성제 혼합물의 정제 키트와 소용돌이와 결합. </li></ol><table border="0" cellpadding="0" cellspacing="0" width="368"><tbody><tr><td> 시약 </td><td> 1X 볼륨 (μL) </td></tr><tr><td> Herculase II 퓨전 DNA 중합 효소 </td><td> 0.5 </td></tr><tr><td> 5 배 Herculase II 반응 버퍼 </td><td> 10 </td></tr><tr><td> 의 dNTP (10 mM의 각) </td><td> 1.25 </td></tr><tr><td> BSA (20 ㎎ / ㎖) </td><td> 1.25 </td></tr><tr><td> 프라이머 MPRA_SfiI_F (25 μM) </td><td> 0.25 </td></tr><tr><td> 프라이머 MPRA_SfiI_R (25 μM) </td><td> 0.25 </td></tr><tr><td> OLS 템플릿 (1-10 attomol) </td><td> 변화 </td></tr><tr><td> 핵산 분해 효소가없는 물 </td><td> 50 </td></tr></tbody></table>ve_content &quot;&gt; 표 2 에멀젼 PCR 반응 믹스 (수상). <ol start="9"><li> 생성물의 외관없이 증폭 생성물의 최대 수율을 제공 농도를 사용하여 (섹션 1.12 참조). </li><li> PCR 튜브에 생성 된 에멀젼을 분배하고 95 ° C에서 (2 분 PCR의 20주기를 수행, 20 × 95 ° C에서 20 초, 55 ° C, 72 ° C에서 30 초에서 20 초], 95시 3 분 ° C). </li><li> 풀 텍싱 간단히 1 ㎖의 이소 부탄올을 첨가하여 각각의 350 μL 에멀젼 PCR 반응을 중단 한 후 DNA 정제 컬럼을 사용하여 증폭 산물을 정제하고. </li><li> 유물없는 증폭 (그림 2B)를 확인하기 위해 2 % 또는 4 % 아가로 오스 겔에 나누어를 실행합니다. </li></ol><img alt="그림이" src="/files/ftp_upload/51719/51719fig2highres.jpg" /> 올리고 뉴클레오티드 합성 라이브러리의 그림 2 준비.) </strong이&gt; 세 가지 다른 원료 올리고 뉴클레오티드 합성 라이브러리 (OLS)은 10 % TBE-우레아 폴리 아크릴 아마이드 젤을 변성에서 실행됩니다. 전체 길이 올리고 뉴클레오티드 (*)에 해당하는 밴드 시각화 라이브러리 1에서 절제과 2 도서관 3 페이지 정화 방해 오염 물질을 포함 할 수 있습니다. 이러한 경우, 아가로 오스 겔상에서 동일한 올리고 뉴클레오티드 라이브러리 런의 개방 및 에멀젼 PCR 증폭 PCR 증폭. B)로 직접 이동 제품. 복잡한 올리고 뉴클레오티드 라이브러리의 PCR 증폭 자주 키메라 제품과 상한 및 하한 밴드로 나타날 수 있습니다 다른 아티팩트를 생성합니다. 에멀젼 PCR은 이러한 아티팩트를 최소화 할 수 있습니다. 2 라이브러리 생성 <ol><li> , 1 % 아가 로스 겔상에서 실행 2 시간, 50 ° C에서 SfiI으로 분해 벡터 pMPRA1 의해 선형화 된 플라스미드 백본을 준비한다 (이 경우, 2.5 KB 단위) 백본 밴드를 절제하고, 겔 정제 스핀 컬럼을 사용하여 정제. </li><li> 다이제스트2 시간 동안 50 ° C에서 SfiI와 단계 1.4에서 에멀젼 PCR 제품과는 DNA 정제 컬럼을 사용하여 정제. </li><li> , 선형화 된 벡터 백본에 프로모터 태그 라이브러리를 결찰하기 위해 소화 PCR 제품의 100 NG, 선형화 된 벡터 백본 및 T4 DNA 리가 아제의 50 NG를 포함하는 반응을 설정합니다. 20 분 리가을 비활성화하는 65 ° C에서 다음 열을 16 ° C에서 밤새 품어. </li><li> E. 변환 연결 반응으로 대장균. 라이브러리 복잡성을 유지하기 위해, 설계 라이브러리에있는 개별의 프로모터 태그 조합이보다 적어도 10 배 더 CFU를 획득하는 것을 목표로하고 있습니다. </li><li> 태그의 총 개수가 약 10 인 경우, 타겟은 전형적으로 도서관 CFU 당 6-8 병렬 변환을 수행함으로써 달성 될 수있다. </li><li> 각 변환의 경우, E. electrocompetent 50 μl를 결합 얼음에 결찰 믹스의 2 μL와 대장균 세포. 800 μL의 SOC에 세포를 전기 천공에 의해 변형 복구병렬 변환에서 세포를 결합 후 1 시간 동안 37 ° C에서 흔들어, 그리고에 의해 매체. </li><li> 변환 효율을 평가하기 위해 50 ~ 100 μg / ml의 카르 베니 실린와 LB 아가 플레이트에 복구 된 세포의 alinquot에서 순차적으로 희석 플레이트 하룻밤에 37 ° C를 품어. / V, V 복구 세포와 V의 전체 볼륨 시리얼 희석 걸리는 나누어의 볼륨 - 총 CFU로 (관찰 CFU) × (희석 계수) × (V V)를 추정한다. </li><li> 동시에, 회수 된 세포의 나머지가 100 μg / ml의 카르 베니 실린이 보충 된 200 ㎖의 LB에 접종하는 데 사용. 진탕 배양기에서 하룻밤 37 ° C에서 세포를 성장하고 표준 절차에 따라 플라스미드 DNA를 분리. </li><li> 2 시간 동안 50 ° C에서 SfiI으로 절연 플라스미드 라이브러리의 분취 다이제스트와 삽입물의 존재를 확인하는 1 % 아가 로스 겔상에서 실행. </li><li> C에 의해 플라스미드 라이브러리를 선형화를 KpnI과 XbaI로를 사용하여 프로모터 변종와 태그 사이에 utting. 소화 효율을 극대화하기 위해, 일련 digestions를 수행 </li><li> 첫째, 1 시간 동안 37 ° C에서 KpnI에서 다이제스트 및 자기 구슬을 사용하여 정제. 둘째, 2 시간 동안 37 ° C에서 1 U 새우 알칼리 포스 파타 아제의 첨가를 XbaI으로 소화, 열은 5 분 동안 65 ° C에서 비활성화 한 다음 자석 구슬을 사용하여 정제. </li><li> 완전한 선형화를 확인하는 1 % 아가 로스 겔상에서 분취 액을 실행. 미가공 플라스미드가 보이면, 선형화 된 단편을 겔 정제한다. </li><li> 포유 동물 세포에 형질 전환에 적합한 MPRA 라이브러리를 생성하기 위해, 선형화 된 중간 라이브러리에 KpnI으로 절단 / XbaI으로 호환 끝으로 ORF를 결찰. </li><li> pMPRAdonor1에서 호환 luc2 ORF 부분을 준비하려면 1 시간 동안 37 ° C에서를 KpnI과 XbaI으로이 플라스미드를 소화하고 1 % 아가 로스 젤에서 실행됩니다. ORF 부분을 절제 (1.7 K이 경우, b) 및 겔 정제 스핀 컬럼을 사용하여 정제. </li><li> 단계 2.3-2.8에서 설명한 바와 같이 선형화 된 라이브러리에 중간 ORF 단편을 복제. </li><li> 1 시간 동안 37 ° C에서 KpnI으로 절단과 MPRA 라이브러리 (1-2 μg에 해당) 나누어지는 다이제스트와 1 % 아가 로스 젤에서 실행됩니다. 소화 라이브러리가 하나의 밴드로 실행하면 추가 밴드가 (일반적으로 중간 구조의 이월에 해당), 라이브러리는 다음과 같이 추가로 정제 할 수있다 관찰하는 경우, 섹션 3으로 진행합니다 : </li><li> 1 시간 동안 37 ° C에서 KpnI으로 절단으로 오염 된 라이브러리의 3-5 μg을 소화하고 4 ℃에서 하룻밤 0.8 % 아가로 오스 겔에서 실행됩니다. </li><li> 올바른 라이브러리 밴드를 절제 겔 정제 스핀 컬럼을 사용하여 DNA를 정제하고, 1 시간 동안 37 ° C에서 고농도의 T4 DNA 리가 아제를 이용하여자가 결찰을 수행한다. 단계 2.8에 설명 된대로 그런 다음 변환 및 최종 라이브러리 DNA 분리를 반복합니다. </li></ol> 3 Transfection, 동요 및 RNA 분리 <ol><li> 각각의 독립적 인 형질 감염, 적절한 배양 배지에서 세포를 필요 매수 (MPRA 라이브러리의 복잡성에 의해 결정된 바와 같이, 논의 참조). 예를 들어, DMEM에서 배양 HEK293T / 17 세포를 10 % FBS 및 L-글루타민 / 페니실린 / 스트렙토 마이신이 보충. 각 라이브러리 및 실험 조건에 대한 적어도 두 개의 독립적 인 형질 감염을위한 세포를 배양. </li><li> MPRA 플라스미드와 함께 배양 된 세포를 형질. 형질 전환 방법 및 조건은 각 세포 유형에 맞게 최적화해야합니다. 각 형질 샘플 들어, 정합 제어로서 플라스미드 DNA의 분취 액 (50-100 NG)를 유지한다. </li><li> 예를 들어, 0.5 × 10 7 HEK293T / 성장 17 세포를 형질에 ~ 나는 30 ㎕의 리포 펙 타민 LTX 10 μL 플러스 시약을 사용하여 혈청 중 감소 된 1 ml의 옵티-MEM에서 10 μg 플라스미드 DNA와 10cm 배양 접시에 50 %의 합류. 5시간 후 형질 전환 혼합물을 제거하고 허용세포는 24 ~ 48 시간 동안 복구 할 수 있습니다. </li><li> 선택적으로, 설계 라이브러리의 상황에 또는 신호에 의존하는 조절 서열을 활성화하는 데 필요한 교란을 수행합니다. </li><li> 세포를 수확과 제조업체의 지침을 사용하여 표준 올리고 (DT) 셀룰로오스 열 또는 구슬을 사용하여 폴리 (A) + mRNA를을 격리 할 것. </li><li> 열 또는 구슬의 최대 결합 용량이 수확 한 세포에서 mRNA의 예상 총 금액을 초과 할 수 있는지 확인합니다. 예를 들어, 3.3에서 기대 수익률은 약 0.5 내지 2.5 μg의 mRNA이다. </li></ol> 4 태그-SEQ <ol><li> 형질 세포 용 해물에서 벡터 DNA의 이월을 제거하기 위해 2.4 ㎕의 터보 DNase의 불 활성화 시약을 1 시간 동안 37 ° C에서 1 μL 터보 DNase의 (2 U) 및 2.3 ㎕의 10 배 터보 DNase의 버퍼 각 20 μL mRNA의 샘플을 처리 추가 RT 후 5 분간 혼합과 90 초간 원심 분리기에 10,000그램 및 위해 신선한 용액 튜브에 옮긴다. </li><li> 확인<html> 아가로 오스 겔상에서의 제품을 각각의 mRNA 시료 60-100 ng의 섹션 4.6에 설명 된 바와 같이 PCR을 수행하고 실행하여 순도. (luc2와 pMPRA1를 사용하는 경우 0.25 킬로바이트) 특정 증폭 산물을 볼 수있는 경우, 열은 치료의 mRNA를 정제 한 후 DNase를 처리를 반복합니다. </li><li> , 태그-SEQ 시퀀싱 라이브러리를 생성하는 cDNA에 기자의 mRNA를 변환 PCR에 의해 시퀀싱 어댑터를 추가합니다. </li><li> 표 3에 설명 된대로의 mRNA / RT 프라이머 혼합물을 설정합니다. 다음 얼음에 놓고 5 분 동안 65 ° C에서 품어. 표 4에 설명 된대로 병행하여, cDNA 합성 반응을 설정합니다. </li></ol><table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td> 시약 </td><td> 1X 볼륨 (μL) </td></tr><tr><td> mRNA의 샘플 (400-700 NG 전체) </td><td> 8 </td></tr><tr><td> 올리고 0dT (50 μM) </td><td> 1 </td></tr><tr><td> 의 dNTP (10 mM의 각) </td><td> 1 </td></tr></tbody></table>cDNA 합성을위한 ove_content &quot;&gt; 표 3 RNA / 역전사 프라이머 믹스. <table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td> 시약 </td><td> 1X 볼륨 (μL) </td></tr><tr><td> 10 배 첨자 III RT 버퍼 </td><td> 이 </td></tr><tr><td> MgCl2를 (25 밀리미터) </td><td> 4 </td></tr><tr><td> DTT (0.1 M) </td><td> 이 </td></tr><tr><td> RNaseOut (40 U / μL) </td><td> 1 </td></tr><tr><td> 첨자 III (200 U / μL) </td><td> 1 </td></tr></tbody></table> 표 4의 cDNA 합성 반응 믹스. <ol start="5"><li> 조심스럽게 mRNA의 / RT 프라이머 cDNA 합성 믹스와 혼합 결합한다. 50 분, 5 분, 85 ° C, 50 ° C에서 부화 한 후 얼음에 배치합니다. 마지막으로, 20 분 동안 37 ° C에서 2 U 리보 뉴 클레아 제의 H와 부화. </li><li> 4-6 μL와 표 5에 설명 된대로 PCR 반응을 설정cDNA의 반응 혼합물 또는 템플릿으로 50 ng의 리포터 플라스미드. 그런 다음 PCR 증폭 (2 분 95 ° C, 26 X [30 초 동안 95 ° C, 30 초 30 초 동안 55 ° C, 72 ° C], 3 분 동안 72 ° C)를 수행합니다. </li></ol><table border="0" cellpadding="0" cellspacing="0" width="428"><tbody><tr><td> 시약 </td><td> 1X 볼륨 (μL) </td></tr><tr><td> 배 PfuUltra II HotStart PCR 마스터 믹스 </td><td> 25 </td></tr><tr><td> 프라이머 TagSeq_P1 (25 μM) </td><td> 0.5 </td></tr><tr><td> 프라이머 TagSeq_P2 (25 μM) </td><td> 0.5 </td></tr><tr><td> 템플릿 (mRNA를 cDNA를 혼합 또는 플라스미드 DNA) </td><td> 변화 </td></tr><tr><td> 핵산 분해 효소가없는 물 </td><td> 50 </td></tr></tbody></table> 표 5 태그-SEQ PCR 반응 혼합물. <ol start="7"><li> 2 % 아가 로스 젤 PCR 제품을 실행 태그-SEQ 라이브러리 증폭 산물에 해당하는 소비세 밴드 (0.25 킬로바이트 사용하는 경우luc2와 pMPRA1) 및 이들의 겔 정화 스핀 열을 정화. </li><li> 수영장, 변성 및 Illumina의 시퀀싱 기기와 직접 정제 된 태그-SEQ 증폭 산물을 시퀀싱. </li><li> 필터 저품질는 시퀀스 태그 내에 30 개 미만의 점수 또는 b) 정확하게 설계된 태그가 일치하지 않는 Phred 품질로 하나 이상의 위치를​​ 포함) 모두를 제거하여 판독한다. 나머지 각 태그는 각 도서관에 나타나는 횟수를 계산합니다. TPM에 (백만 시퀀싱 태그 당 태그) 태그 카운트를 정규화 한 후 시퀀싱 라이브러리의 각 쌍에 대한 플라스미드 유래 태그 카운트 위에 유래의 mRNA 태그 카운트의 비율을 계산한다. 서로 다른 여러 태그를 각 시퀀스 변종에 연결 한 경우, 다운 스트림 분석을 위해 자신의 평균 비율을 사용합니다. </li></ol>

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저자는 그들이 더 경쟁 금전적 이해 관계가 없다고 선언합니다.

MPRA is a flexible and powerful tool for dissection of sequence-activity relationships in gene regulatory elements. The success of MPRA experiments depend on at least three factors: 1) careful design of the sequence library, 2) minimization of artifacts during amplification and cloning, and 3) high transfection efficiency. 
The possible lengths of the variable regions in the reporter constructs are largely determined by the synthesis or cloning technology used. Standard OLS is generally limited to about 200 nt, but this protocol is compatible with inserts up to at least 1,000 nt. Note that variable regions that are highly repetitive or contain strong secondary structures may end up underrepresented due to PCR and cloning biases. The length of the tags that identify each of the variable regions should be 10-20 nt and the collection of tags should ideally be designed such there are at least two nucleotide differences between any pair. Tags that contain the seed sequences of known microRNA or other factors that might influence mRNA stability should also be avoided when possible.
A key parameter in the design of MPRA experiments is the total number of distinct reporter constructs to be included in the library (the design complexity, denoted CD). In practice, CD is limited by the number of cultured cells that can be transfected. As a rule of thumb, the total number of transfected cells should be at least 50-100 times greater than CD. For example, if 20 million cells can be transfected with a transfection efficiency of 50%, then CD should be no more than ~200,000. Note that CD is equal to the number of distinct regulatory sequence variants multiplied by the number of distinct tags per sequence. The more distinct tags are linked to each regulatory sequence, the more accurate the estimate of the activity of that sequence can be made (because measurements from distinct tags can be averaged), but the fewer distinct variants can be assayed in one experiment. The optimal choice depends on the experimental design. In a simple &#8220;promoter bashing&#8221; experiment, where a mathematical model will be fitted to the aggregated measurements, a single tag per variant is usually sufficient. In a screen for single-nucleotide polymorphisms that cause changes in regulatory activities, it may be necessary to use 20 or more tags per allele to obtain statistically robust results, because comparing each pair of alleles requires a separate hypothesis test. 
If the sequences to be assayed are not expected to contain transcription start sites, a constant promoter can also be added in the same fragment. For example, pMPRAdonor2 (Addgene ID 49353) includes a minimal TATA-box promoter that is useful when the upstream variable region is expected to have significant enhancer activity, while pMPRAdonor3 (Addgene ID 49354) includes a modified, strong SV40 viral promoter that is useful when the variable region is expected to contain silencer activity or other negative regulatory elements. 
Raw OLS products often contain a significant fraction of truncated oligonucleotides. These may interfere with accurate PCR amplification of the designed sequences, particularly when there is significant homology between them. Using PAGE purification to remove truncated synthesis products and emulsion PCR to minimize amplification artifacts are effective techniques for ensuring high library quality. If either step is impractical, it is imperative to minimize the number of PCR cycles used at each amplification step. Selection and expansion of the cloned library in liquid culture is generally sufficient to maintain the design complexity, but if recombination-prone vectors are to be used or significant representation bias is observed, the recovered cells can instead be plated directly onto large LB agar plates, expanded as individual colonies and then scraped off for DNA isolation. It is also important to consider the potential impact of synthesis errors, which are typically found at a rate of 1:100-500 in OLS. Full-length sequencing of the reporter constructs prior to transfection is recommended to identify and correct for such errors.
It is not necessary to introduce reporter constructs into every cell in the transfected culture, but transfection efficiencies below ~50% may lead to poor signal to noise ratios. It is advisable to optimize transfection conditions prior to performing MPRA experiments in a new cell type. When working with hard-to-transfect cell types, MPRA signals can be boosted by pre-selecting transfected cells. The pMPRA vector series includes variants that constitutively express a truncated cell surface marker that can be used to physically enrich for transfected cells prior to RNA isolation (for example, Addgene IDs 49350 and 49351).

A correction to the author list has been made for article <a href="http://www.jove.com/video/51719">Massively Parallel Reporter Assays in Cultured Mammalian Cells</a>.

Erratum: Massively Parallel Reporter Assays in Cultured Mammalian Cells

대규모 병렬 리포터 분석 실험은 (MPRA)는 DNA 서열 1 (7)을 수천 수백 수천의 전사 규제 활동의 다중 측정을 할 수 있습니다. 자신의 가장 일반적인 실시 예에서, 다중화는 오픈 리딩 프레임의 하류 서열 식별 태그가 포함되어 합성 리포터 유전자를 관심있는 각각의 시퀀스를 연결함으로써 달성된다 (ORF를,도 1). 형질 감염, RNA 분리 및 리포터 유전자 전 사체의 3 &#39;말단의 깊이 순서화에 따라, 결합 된 서열의 상대적인 활동은 식별 태그의 상대적인 풍부로부터 추론 될 수있다. <img alt="그림 1" src="/files/ftp_upload/51719/51719fig1highres.jpg" /> MPRA의 그림 1 개요. MPRA 기자의 라이브러리는 합성으로 추정 조절 서열을 결합하여 구성되어 구축서열 식별 태그 뒤에 (예로서 루시페라아제 또는 GFP) &quot;불활성&quot;ORF로 구성 리포터 유전자. 도서관은 배양 된 세포의 인구에 대거 형질 전환하고, 전사 리포터 mRNA를 연속적으로 회수한다. 깊은 순서는 기자의 mRNA와 형질 전환 된 플라스미드 중 각 태그의 발생 수를 계산하는 데 사용됩니다. 플라스미드 카운트 대응하는 조절 서열의 활성을 추정하는 데 사용될 수있는 동안의 mRNA의 비를 계산. 멜린 니코의 허가를 적응, 등 2. MPRA은 관심 궤적 걸쳐 신규 조절 요소에 대해 개별 유전자 조절 요소는 1) 광범위한 돌연변이 유발, 2) 주사를 포함하여, 실험 다양한 설계에 적용 할 수 있으며, 3) 추정의 세트 천연 유전 적 변이의 효과를 테스트 프로모터, 인핸서 또는 소음기, 합성 규제 요소 사) 반 합리적인 엔지니어링. 리시퀀스 변종 braries은 퇴화 올리고 1,4, 조합 결찰 8 게놈 DNA 5의 조각화 올리고 뉴클레오티드 라이브러리 프로그램 마이크로 어레이 2,3,6,7-에 합성 (OLS), 조립 등 다양한 방법을 사용하여 생성 할 수 있습니다. 이 프로토콜은 프로모터의 라이브러리의 구조를 설명 변종 OLS와 pMPRA1 및 pMPRAdonor1 벡터 (각각 Addgene ID를 49349 및 49352; http://www.addgene.org)를 사용하여, 배양 된 포유류 세포 및 후속 정량에이 라이브러리의 일시 발현을 관련 태그의 깊은 시퀀싱 (태그-SEQ)에 의해 프로모터 활동. 이 프로토콜의 이전 버전 (2013) 연구 23, 800-811 멜린 니코 등. 자연 생명 공학 30, 271-277 (2012)과 Kheradpour 등. 게놈에보고 된 연구에 사용되었다.

<table><tbody><tr><td>Oligonucleotide library synthesis</td><td>Agilent, CustomArray, or other OLS vendors</td><td>custom</td><td>If using OLS construction method</td></tr><tr><td>pMPRA1</td><td>Addgene</td><td>49349</td><td>MPRA plasmid backbone</td></tr><tr><td>pMPRAdonor1</td><td>Addgene</td><td>49352</td><td>luc2 ORF donor plasmid</td></tr><tr><td>TE 0.1 Buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0)</td><td>Generic</td><td>n/a</td><td>OLS buffer</td></tr><tr><td>Novex TBE-Urea Gels, 10%</td><td>Life Technologies</td><td>EC6875BOX</td><td>PAGE purification of OLS products</td></tr><tr><td>Novex TBA-Urea Sample Buffer</td><td>Life Technologies</td><td>LC6876</td><td>PAGE purification of OLS products</td></tr><tr><td>SYBR Gold Nucleic Acid Gel Stain</td><td>Life Technologies</td><td>S-11494</td><td>PAGE purification of OLS products</td></tr><tr><td>Micellula DNA Emulsion &amp;amp; Purification Kit</td><td>EURx/CHIMERx</td><td>3600-01</td><td>Library amplification by emulsion PCR</td></tr><tr><td>Herculase II Fusion DNA Polymerase</td><td>Agilent</td><td>600675</td><td>Polymerase for emulsion PCR</td></tr><tr><td>SfiI</td><td>New England Biolabs</td><td>R0123S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>KpnI-HF</td><td>New England Biolabs</td><td>R3142S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>XbaI</td><td>New England Biolabs</td><td>R0145S</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>T4 DNA Ligase (2,000,000 units/ml)</td><td>New England Biolabs</td><td>M0202T</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>One Shot TOP10 Electrocomp &lt;em&gt;E. coli&lt;/em&gt;</td><td>Life Technologies</td><td>C4040-50</td><td>Library cloning with pMPRA vectors</td></tr><tr><td>LB agar and liquid media with carbenicllin</td><td>Generic</td><td>n/a</td><td>Growth media for cloning</td></tr><tr><td>E-Gel EX Gels 1%</td><td>Life Technologies</td><td>G4010-01</td><td>Library verification and purification</td></tr><tr><td>E-Gel EX Gels, 2%</td><td>Life Technologies</td><td>G4010-02</td><td>Library verification and purification</td></tr><tr><td>MinElute Gel Extraction Kit</td><td>Qiagen</td><td>28604</td><td>Library and backbone purification</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Library DNA isolation</td></tr><tr><td>Cell culture media</td><td>n/a</td><td>n/a</td><td>Experiment-specific</td></tr><tr><td>Transfection reagents</td><td>n/a</td><td>n/a</td><td>Experiment-specific</td></tr><tr><td>MicroPoly(A)Purist Kit</td><td>Life Technologies</td><td>AM1919</td><td>mRNA isolation</td></tr><tr><td>TURBO DNA-free Kit</td><td>Life Technologies</td><td>AM1907</td><td>Plasmid DNA removal</td></tr><tr><td>SuperScript III First-Strand Synthesis System</td><td>Life Technologies</td><td>18080-051</td><td>cDNA synthesis</td></tr><tr><td>PfuUltra II Hotstart PCR Master Mix</td><td>Agilent</td><td>600850</td><td>Polymerase for Tag-Seq PCR</td></tr><tr><td>Primers (see text)</td><td>IDT</td><td>custom</td><td>PAGE purify Tag-Seq primers</td></tr></tbody></table>

massively parallel reporter assays in cultured mammalian cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

MPRA 고해상도, 전사 조절 요소의 시퀀스 - 활성 관계를 정량적으로 절개를 용이하게한다. 성공적인 MPRA 실험은 전형적으로 형질 라이브러리 (도 3a)에서 서열의 대부분 고도로 재현 가능한 측정 값을 수득한다. 나쁨 재현성 (도 3b)이 관찰되는 경우, 이는 정량 서열 중 하나) 낮은 절대 활성, 또는 2) 낮은 형질 감염 효율 하나에, 회수 된 RNA 샘플에서 리포터의 mRNA 너무 낮은 농도를 나타낸다. 도 4는 시금 생성 대표적인 &quot;공간 정보&quot;를 보여준다 ~ 1,2 또는 센다이 바이러스에의 노출없이 HEK293 세포에서 인간 IFNB 유전자의 상류에 145 bp의 서열의 임의의 변형 37000. 발기인 TATA 박스 알려진 근위 증강 (10)는 명확하게 정보가 풍부한 REGI으로 식별 할 수 있습니다바이러스 의존적으로 기능. <img alt="그림 3" src="/files/ftp_upload/51719/51719fig3highres.jpg" /> 그림 3 태그-SEQ 재현성. 고 (A)과 낮은 (B) 재현성이 독립적 인 복제 형질에서 태그-SEQ 데이터의 예를 나타내는 산포도. 후자의 플롯은 반복에의 한 높은 mRNA의 수가 많은 아웃 라이어의 태그를 보여줍니다. 이러한 유물은 일반적으로 기자의 mRNA의 농도 인해 기자의 구조 중 낮은 절대 활동, 또는 낮은 형질 전환 효율에 하나, 정량적 PCR 증폭 너무 낮았다을 나타냅니다. <img alt="그림 4" src="/files/ftp_upload/51719/51719fig4highres.jpg" /> 인간의 IFNB 전사 시작 사이트와 근위 증강의 그림 4 정보 배출량 측정. </str옹&gt; 상류 IFNB 인간 유전자의 뉴클레오티드 145 (NT) 영역의 약 37,000 랜덤 변형 (A)로 및 센다이 바이러스 (B)의 노출없이 HEK293 세포를 사용 MPRA 분석 하였다. 파란색 막대는 각 위치에서 리포터 출력 및 뉴클레오타이드 간의 상호 정보를 나타낸다. 근위 증강 및 TATA 박스는 바이러스 감염시 높은 정보 콘텐츠의 영역으로 눈에 띄는.

Watch this Scientific Journal Video about Massively Parallel Reporter Assays in Cultured Mammalian Cells at JoVE.com

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

배양 된 포유 동물 세포에서 대규모 병렬 리포터 분석 실험

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

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21.5K 조회수.  Broad Institute. The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

비디오: Massively Parallel Reporter Assays in Cultured Mammalian Cells

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in vitro analysis are well suited to study of large numbers of cells (&ge;105) in milliliter-scale volumes (&ge;0.1 ml). However, there are many instances in which it is necessary or desirable to scale down culture size to reduce consumption of the cells of interest and/or reagents required for their culture, stimulation, or processing. Unfortunately, conventional approaches do not support precise and reproducible manipulation of micro-scale cultures, and the microfluidics-based automated systems currently available are too complex and specialized for routine use by most laboratories. To address this problem, we have developed a simple and versatile technology platform for automated culture, stimulation, and recovery of small populations of cells (100 - 2,000 cells) in micro-scale volumes (1 - 20 &mu;l). The platform consists of a set of fibronectin-coated microcapillaries ("cell perfusion chambers"), within which micro-scale cultures are established, maintained, and stimulated; a digital microfluidics (DMF) device outfitted with "transfer" microcapillaries ("central hub"), which routes cells and reagents to and from the perfusion chambers; a high-precision syringe pump, which powers transport of materials between the perfusion chambers and the central hub; and an electronic interface that provides control over transport of materials, which is coordinated and automated via pre-determined scripts. As an example, we used the platform to facilitate study of transcriptional responses elicited in immune cells upon challenge with bacteria. Use of the platform enabled us to reduce consumption of cells and reagents, minimize experiment-to-experiment variability, and re-direct hands-on labor. Given the advantages that it confers, as well as its accessibility and versatility, our platform should find use in a wide variety of laboratories and applications, and prove especially useful in facilitating analysis of cells and stimuli that are available in only limited quantities.

We have developed an automated cell culture and interrogation platform for micro-scale cell stimulation experiments. The platform offers simple, versatile, and precise control in cultivating and stimulating small populations of cells, and recovering lysates for molecular analyses. The platform is well suited to studies that use precious cells and/or reagents.

마이크로 스케일 세포 자극 실험을위한 다목적 자동화 플랫폼

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

사진-가교 매핑 및 라이브 포유류 세포에서 Bioorthogonal 화학 GPCRs에 프로브 화학의 유전 결합을 최적화

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

화학

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

의학

A Microscopic Phenotypic Assay for the Quantification of Intracellular Mycobacteria Adapted for High-throughput/High-content Screening

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since several decades, the sudden burst of multi- and extensively-drug resistant strains is a serious threat for the control of tuberculosis. Therefore, it is essential to identify new targets and pathways critical for the causative agent of the tuberculosis, Mycobacterium tuberculosis (Mtb) and to search for novel chemicals that could become TB drugs. One approach is to set&#160;up methods suitable for the genetic and chemical screens of large scale libraries enabling the search of a needle in a haystack. To this end, we developed a phenotypic assay relying on the detection of fluorescently labeled Mtb within fluorescently labeled host&#160;cells using automated confocal microscopy. This in vitro assay allows an image based quantification of the colonization process of Mtb&#160;into the host and was optimized for the 384-well microplate format, which is proper for screens of siRNA-, chemical compound- or Mtb mutant-libraries. The images are then processed for multiparametric analysis, which provides read&#160;out inferring on the pathogenesis of Mtb&#160;within host cells.

Here, we describe a phenotypic assay applicable to the High-throughput/High-content screens of small-interfering synthetic RNA (siRNA), chemical compound, and Mycobacterium tuberculosis mutant libraries. This method relies on the detection of fluorescently labeled Mycobacterium tuberculosis within fluorescently labeled host&#160;cell using automated confocal microscopy.

고처리량/고함량 스크리닝에 적용된 세포내 진균의 정량화를 위한 현미경 표티픽 분석

Despite the availability of therapy and vaccine, tuberculosis (TB) remains one of the most deadly and widespread bacterial infections in the world. Since ...

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A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

포유류 세포에서 인트로닉 마이크로RNA 성숙을 분석하기 위한 리포터 분석

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

암 연구학

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1st generation assay) or fluorescent protein reporters (2nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solution-based readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

A high throughput, real-time assay was developed to simultaneously identify (1) eukaryotic cell-penetrant antimicrobials targeting an intracellular bacterial pathogen, and (2) assess eukaryotic cell cytotoxicity. A variation on the same technology was thereafter combined with digital dispensing technology to enable facile, high-resolution, dose-response, and two- and three-dimensional synergy studies.

높은 처리량, 실시간, 세포 내 항균 활동의 이중 판독 테스트 및 진핵 세포의 세포 독성

Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

형광 현미경으로 여러 세대 이상 라이브 포유류의 세포에 플라스미드 복제 모니터링

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

생물학

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Techniques for generating a library of short peptides that can activate mast cells via the MRGPRX2 receptor are described. Associated techniques are easy, inexpensive, and can be extended to other cell receptors.

엔지니어링 된 HEK 세포를 사용하여 MRGPRX2를 활성화하는 펩티드 스크리닝

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

세포 독성의 지속적인 실시간 모니터링을위한 자율적으로 바이오 루미 네 센트 포유 동물 세포

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...