이 작품은 NIH 감독의 새로운 혁신 상 프로그램 (ED)에서 부여에 의해 지원되었다. AA는 국립 암 연구소 (National Cancer Institute)와 국립 과학 재단 (National Science Foundation)에서 학부 상에 의해 부분적으로 지원되었다.

1. 바이러스 성 벡터를 만들기 목적 유전자뿐만 아니라, 양성 및 음성 대조군에 대한 코딩 서열은, 전통적인 클로닝 방법 (PCR 증폭, 제한 효소 및 라이 게이션)에 의하여 pBABEpuro 삽입된다. BamHI로, SnaBI로, EcoRI 및 SalI로 : DNA가 삽입 될 수있다 벡터 네 개의 제한 부위가있다. <ol><li> QIAGEN 플라스미드 미디 키트를 사용하여 유능한 세포에서 형질 급 플라스미드 DNA를 준비합니다. </li><li> NanoDrop2000 분광 광도계를 이용하여 DNA 농도를 측정한다. </li></ol> 2. 레트로 바이러스 생산 Plat의 E-패키징 세포주 NIH 3T3 세포가 관심 cDNA를 제공한다 ecotropic 레트로 바이러스를 생산하는 데 사용한다. <ol><li> 냉동 세포에서 Plat의-E 문화 구축 <ol><li> 빠르게 37 ° C의 물을 욕조에 Plat의-E 세포를 해동하고 15 ML 원뿔 튜브에 전송할 수 있습니다. 천천히 (뒤를 Plat의-E 매체의 9를 가하여lbecco 개변 이글 DMEM 배지 + 10 % 소 태아 혈청 (FBS) 및 스트렙토 마이신 +). </li><li> 180 X에서 튜브를 원심 분리기 g 5 분 상층 액을 버린다. </li><li> 10cm의 배양 접시에 작은지면-E 매체 및 전송 10 mL로 세포를 다시 일시 중지합니다. </li><li> 그들은 80 ~ 90 %의 합류 (약 2 일) 때까지 37 ° C, 5 % CO 2 배양기에서 세포를 품어. </li></ol></li><li> 셀 분할 <ol><li> 매체를 대기음 PBS로 한 번 세포를 씻으십시오. </li><li> 0.05 % 트립신 EDTA 2 ㎖를 추가하고 실온에서 1 분 동안 품어. </li><li> 도청 손가락으로 세포를 분리 Plat의-E 배지 10 mL를 넣고 15 ㎖의 튜브에 세포 현탁액을 전송. </li><li> 180 X에서 튜브를 원심 분리기 g 5 분 상층 액을 버린다. </li><li> 작은지면-E 매체의 10 ml의 세포를 재현 탁 1에서 새로운 요리를 시드 : 4-1 : 6 희석. </li></ol></li><li> 작은지면-E 시드 및 형질 <ol><li> 시드 2 × 10 (6)어떤 항생제없이 작은지면-E 매체를 이용하여 10cm 배양 접시 당 세포. </li><li> 37 ° C, 5 % CO 2 배양기에서 세포 O / N을 품어. </li><li> 다음 날, 1.5 ml의 미세 원심 분리 튜브에 옵티-MEM 300 μl를 전송합니다. </li><li> 옵티-MEM으로 준비 관, 폴리에틸렌 이민의 27 μL (PEI), 비용 효율적인 형질 전환 시약 (12)를 추가합니다. 도청 손가락으로 부드럽게 섞어 실온에서 5 분 동안 품어. </li><li> , 옵티-MEM / PEI 튜브에 형질 급 플라스미드 DNA의 9 μg의 추가 텍싱하여 부드럽게 혼합하고, 실온에서 15 분 동안 품어. </li><li> 추가 DNA / PEI Plat의-E 접시에, 37 ° C에서 O / N을 품어 복합 한 방울 씩, 5 % CO 2. </li><li> 다음 날, 형질 전환 시약을 포함하는 매체를 대기음 (항생제없이) 신선한 Plat의-E의 10 ml의 배지를 추가합니다. </li><li> 인큐베이터에 세포를 돌려줍니다. </li></ol></li></ol> 3. NIH 3T3 세포 및 감염 <ol><li> NIH 구축3T3 문화 및 시드 <ol><li> 빠르게 37 ° C의 물을 욕조에 NIH 3T3 세포를 해동하고 15 ML 튜브로 전송할 수 있습니다. 천천히 NIH 3T3 배지 (DMEM + 10 % FBS)의 구를 가하여. </li><li> 180 X에서 튜브를 원심 분리기 g 5 분 상층 액을 버린다. </li><li> 10cm 배양 접시 NIH 3T3 매체와 전사 10 mL로 세포를 재현 탁. </li><li> 37 ° C, 5 % CO 2 인큐베이터에서 세포를 배양한다. 그것은 자연 변환 (그리고 초점 형성 따라서 기초 수준)의 주파수로 문화 포화 상태에 도달하면 증가, NIH 3T3 세포는 항상 underconfluent (50~60%)을 유지하는 것이 매우 중요합니다. </li><li> 10cm 접시 당 종자 3 × 10 5 세포는 다음날 감염 O / N을 배양한다. </li></ol></li><li> 감염 <ol><li> 0.45 ㎛의 세공 크기를 나일론 멤브레인 필터로 여과 한, 10 ㎖의 일회용 주사기를 사용하고, 15 ㎖ 중에 전송해서 E-Plat의 접시에서 레트로 바이러스 상등액을 수확튜브. </li><li> (항생제없이) 세포에 신선한 Plat의-E 매체의 10 ML을 추가하고 인큐베이터에 반환. </li><li> 감염 될 수있는 NIH 3T3 세포 접시에서 매체를 대기음, 바이러스 함유 필터링 된 상층 액의 5 정기적 인 NIH 3T3 매체 ml의 5 mL를 추가합니다. </li><li> 6 ㎍ / ml의 농도로 폴리 브렌 접시에 추가. </li><li> 37 ° C, 5 % CO 2의 세포 O / N을 품어. </li><li> 감염의 두 번째 라운드에 대한 3.2.5 - 다음 날, 반복 3.2.1 단계를 반복합니다. </li><li> 일반 NIH 3T3 매체와 바이러스 함유 배지를 교체합니다. </li><li> 필요에 따라 매체를 교체, NIH 3T3 세포를 2 ~ 3 주 동안 성장하는 것을 허용합니다. 복제 판에서 전체 세포 용 해물에 기존의 단백질 전기 영동 및 면역에 의해 관심의 단백질의 발현을 확인합니다. </li></ol></li></ol> 4. 염색 및 정량화 <ol><li> 크리스탈 바이올렛 염색 <ol><li> NIH 3T3 매체를 대기음 요리를 배치얼음에. </li><li> 얼음처럼 차가운 PBS로 두 번 요리를 씻으십시오. </li><li> 10 분 동안 빙냉 메탄올로 세포를 고정. </li><li> 얼음에서 요리를 제거하고 메탄올을 대기음, 25 % 메탄올 (RT)에서 만든 0.5 % 크리스탈 바이올렛 용액 3 ㎖를 추가합니다. </li><li> RT에서 5 분 동안 요리를 품어. </li><li> 크리스탈 바이올렛 솔루션을 기음과 컬러가 린스 벗기하지 않을 때까지 조심스럽게 밀리-Q의 H 2 O와 접시를 씻어. </li><li> 요리는 벤치 탑에 O / N을 건조하도록 허용 (발견). </li></ol></li><li> 초점 정량화 <ol><li> 요리가 건조되면, 접시에 세포의 음울 색깔의 컬렉션을 측정하는 통치자를 사용합니다. 만 등의 직경보다 5mm 된 것을 계산 &quot;초점을.&quot;초점의 수를 기록하고 평균과 컨트롤에 비해 중요성을 계산합니다. </li></ol></li></ol>

<ol>
	<li>Clark, G. J., Cox, A. D., Graham, S. M., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+assays+for+Ras+transformation.">Biological assays for Ras transformation.</a> Methods in enzymology. , 255-395 (1995).</li><li>Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell biology : a laboratory handbook. , 345-352 (2006).</li><li>Raptis, L., Vultur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+assays.">Neoplastic transformation assays.</a> Methods in molecular biology. 165, 151-164 (2001).</li><li>Johnson, P. J., Coussens, P. M., Danko, A. V., Shalloway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressed+pp60c-src+can+induce+focus+formation+without+complete+transformation+of+NIH+3T3+cells.">Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.</a> Molecular and cellular biology. 5, 1073-1083 (1985).</li><li>Bonner, T. I., Kerby, S. B., Sutrave, P., Gunnell, M. A., Mark, G., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+biological+activity+of+human+homologs+of+the+raf/mil+oncogene.">Structure and biological activity of human homologs of the raf/mil oncogene.</a> IMolecular and cellular biology. 5, 71400-71407 (1985).</li><li>Yancopoulos, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+can+cooperate+with+ras+to+transform+normal+cells+in+culture.">N-myc can cooperate with ras to transform normal cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 82, 5455-5459 (1985).</li><li>Fujiwara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+activity+of+purinergic+receptor+P2Y,+G+protein+coupled,+8+revealed+by+retroviral+expression+screening.">Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.</a> Leukemia &amp; lymphoma. 48, 978-986 (2007).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene. 7, 1063-1066 (2000).</li><li>Morgenstern, J. P., Land, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+mammalian+gene+transfer:+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line.">Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.</a> Nucleic acids research. 18, 3587-3596 (1990).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397, 3173-3178 (2010).</li><li>Klaver, B., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+5'+and+3'+long+terminal+repeat+promoter+function+in+human+immunodeficiency+virus.">Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus.</a> Journal of virology. 68, 3830-3840 (1994).</li><li>Fukumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+gene+transfection+by+DNA+compaction+at+pH+4.0+using+acidified,+long+shelf-life+polyethylenimine.">Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine.</a> Cytotechnology. 62, 73-82 (2010).</li><li>Fox, E. J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells.">S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells.</a> The Biochemical journal. 359, 361-367 (2001).</li><li>Yun, J. S., Rust, J. M., Ishimaru, T., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+role+of+the+Mad+family+member+Mad3+in+cerebellar+granule+neuron+precursor+proliferation.">A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation.</a> Molecular and cellular biology. 27, 8178-8189 (2007).</li><li>Gore, Y., Lantner, F., Hart, G., Shachar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mad3+negatively+regulates+B+cell+differentiation+in+the+spleen+by+inducing+Id2+expression.">Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression.</a> Molecular biology of the cell. 21, 1864-1871 (2010).</li><li>Barisone, G. A., Yun, J. S., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+cerebellar+proliferation+to+tumorigenesis:+new+insights+into+the+role+of+Mad3.">From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3.</a> Cell cycle. 7, 423-427 (2008).</li><li>Barisone, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+MXD3+in+proliferation+of+DAOY+human+medulloblastoma+cells.">Role of MXD3 in proliferation of DAOY human medulloblastoma cells.</a> PloS One. 7, e38508 (2012).</li><li>Nair, S. K., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-ray+structures+of+Myc-Max+and+Mad-Max+recognizing+DNA.+Molecular+bases+of+regulation+by+proto-oncogenic+transcription+factors.">X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.</a> Cell. 112, 193-205 (2003).</li></ol>

저자가 공개하는 게 없다.

FFA는 체외에서 악성 변화를 평가하기위한 빠르고 쉬운 방법을 제공합니다. 이는 후보 유전자의 상대적으로 큰 수의 검사 의무이며, 그 완만 한 기술적 요구는 비용 효율적인 만든다. 또한, 두 개 이상의 유전자를 공 발현 할 수있다 조합 발암 잠재력을 평가하는 (때때로 &quot;협동&quot;분석법이라 함). 이 분석의 장점은 간단 기술, 정량의 용이성과 상대적으로 짧은 턴어라운드 시간에 의존하...

종양 세포 유전자 발현의 패턴에서 증식 및 형태 학적 변화 후성 유전학에, 변경의 폭 넓은 정상 대조 구별 될 수있다. 후자 중에서, 궁극적으로 동물에 주입시 종양을 형성하는 능력은 악성화 (3)의 유용한 측정 지표 인 혈청에 의존하여, 접점 (밀도) 억제의 상실, 앵커리지 - 독립된 확산 인수를 감소. 생체 외 및 생체 내 분석 몇몇 셀룰러 변환을 위해 개발되었다. 시험 관내 분석법을 목표 (환원 혈청 성장)을 식별하고 배양 모폴로지 (초점 형성 어 세이), 문화 역학 (성장 속도, 포화 농도) 및 성장 인자의 변화가 측정 또는 요구 사항 앵커리지 (소프트 한천의 성장). 세포 유형의 악성 특성을 결정하기위한 금 표준 실험 동물에서 종양 형성 (이종) 남아있다. 그러나, t그는 비용 및 생체 내 연구의 길이는 항상 후보 종양 유전자의 첫 번째 검증 단계 또는 스크리닝 그들을 정당화하지 않습니다. 어떤 시험 관내 분석은 유전자의 발암 가능성의 명확한 평가를 제공하지 않지만, 그들은 생체 내 (in vivo) 연구가 미래를 좁힐 수 발암 가능성에 대한 통찰력을 제공 않습니다. 시험관 내에서 종양 발생 가능성을 평가하기위한 가장 널리 사용되는 시스템 중 하나는 초점 형성 분석법이있다. 이 접근법은 NIH 3T3 마우스 섬유 모세포, 강한 접촉 억제를 나타내고 형질 전환되지 않은 세포주의 사용에 의존한다. 밀도에 의존하는 성장의 손실 종양 유전자 결과의 과발현; 형질 전환 된 세포를 용이하게 형질 전환되지 않은 세포의 배경에 대해 단층 가시화 &quot;초점&quot;을 형성하는, 다층으로 성장할 수있다. 접점의 ON 인 손실에 의해 입증되는 바와 같이 포커스 정량법 형성 후, 악성 변환을 유도하는 후보의 종양 유전자 능력을 측정측정 표현형으로 ition. FFA (예를 들면, Src에 4 BRAF 5) 단백질 키나제의 과발현, 전사 인자 (예, N-myc의 6) (예 P2RY8 7), G는 단백질 - 결합 수용체와 GTP 아제 (예에 의해 변형을 평가하기 위해 사용되어왔다 , 라스 1), 다른 사람의 사이에서. 이 분석의 상대적 용이성은 유전자의 과발현은 체외에서 NIH 3T3 마우스 섬유 아세포를 변환 할만큼 충분한 지 여부에 대한 신속하고 시각적으로 명확한 답을 제공 할 것입니다 좋은 선택입니다. 이 프로토콜에서 설명 FFA 패키징 바이러스 단백질을 제공 Plat의 E-패키징 세포주 (8)를 사용하고, 레트로 바이러스 벡터 pBABEpuro 9 (Addgene 플라스미드 1764)는 레트로 바이러스를 생성한다. 관심의 유전자를 포함하는 구조체 pBABEpuro 형질 감염 후, E-Plat의 세포주는 NIH 3T3 세포를 감염시키기 위해 사용될 수 ecotropic 레트로 바이러스를 생성 할 것이다. 티유전자 전달에있어서의 그의 바이러스 전통적인 화학적 형질 전환 방법보다 더 효율적이며 지속적으로 10 유전자를 발현 할 수있는 방법을 제공한다. 일단 NIH 3T3 세포의 게놈 내로 혼입, 목적 유전자의 과발현은 바이러스 긴 말단 반복 (LTR) 프로모터 (11)에 의해 구동된다. 이 상수는 식 NIH 3T3 세포상의 초점의 형성에 의해 측정 된 관심 유전자, 종양 활성을 가지고 있는지 여부를 결정하는데 사용될 수있다.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td>cell culture media</td>
		</tr>
	</tbody>
</table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3는 MYC / MAX / MAD 네트워크의 구성원 인 염기성 헬릭스 - 루프 - 헬릭스 류신 지퍼 (bHLHZ) 전사 인자이다. 그것은 MAD 가족 13-15의 비정형 부재이며, 그것은 암화 (16, 17)에 관여하는 것으로보고되었다. pBABEpuro (대조군)과 MYC (양성 대조군)과 비교했을 때, MXD3가 과발현 된 NIH 3T3 요리는 훨씬 적은 초점 (그림 1A)를했다. 도 1b의 데이터는 중요성을 결정하기 위해 여러 실험으?...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

초점 형성의 분석은 후보 종양 유전자의 변화 가능성을 평가하기위한 간단한 방법을 제공한다.

형성 초점 : 세포 기반 분석은 유전자의 발암 성 가능성을 파악하기 위해

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.7K 조회수.  University of California, Davis.  초점 형성의 분석은 후보 종양 유전자의 변화 가능성을 평가하기위한 간단한 방법을 제공한다. 

비디오: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

PTSD의, 신경 Hematologic, 그리고 행동 상관 관계를 누설하는 행위에 대해서는 동물 모델에서 생체

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

연구

의학

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

태아 마우스 모델에서 Bioactive 에이전트의 Intratracheal 관리를위한 소설 외과 접근

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

인간 혈청에있는 N-메틸-D-아스파 라진 산염 수용체 또는 도파민 2 수용체에 대한 항체를 검출하는 높은 처리량 유동 세포 계측법 세포 기반 분석

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

수정<em&gt; 시험관</em&gt; 침공 분석은 호르몬, 중재 암 세포 침략의 사이토 카인 및 / 또는 성장 인자의 잠재적 인 역할을 확인하는

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

소설<em&gt; 생체</em&gt; 유전자 전달 기술과<em&gt; 시험관</em근골격계 질환의 뼈 손실의 연구를위한&gt; 셀 기반 분석 실험

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

3 차원 (3D) 종양 회전 타원체 침공 분석

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

소프트 한천 콜로니 형성 분석의 활용은 유방암 세포에서 종양 유발의 억제를 확인하는

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

TRUE 유전자 음소거 : 잠재적 인 암 치료제에 대한 헵 타머 형 소형 가이드 RNA 라이브러리의 심사

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

동계 쥐 흑색 종 세포의 주입은 폐에서 그들의 전이성 잠재력을 확인하는 방법

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

인간의 혈액 - 뇌척수액 장벽의 맥락총 상피 세포 기반 모델은 기저 측면에서 세균 감염을 연구하는

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...