We thank Drs. Alison Heffer and Yong Lu for setting up the microinjection apparatus and sharing their invaluable knowledge and experience with insect RNAi. This work was supported by the National Institutes of Health (R01GM113230 to L.P.).

The authors have nothing to disclose.

정교한 모델 시스템 (마우스, 날아 벌레)의 소수는 20 세기 동안 개발 동안, 21 세기 새로운 동물 시스템의 물결이 전 세계에 걸쳐 연구실에서 개발중인 보았다. 이 새로운 시스템은 과학자 만 &#39;표준&#39;모델 시스템을 사용하여 탐색 할 수 없습니다 비교, 진화의 문제를 해결 할 수 있습니다. 새로운 모델이 배포 실험실 배양, 유전자 식별 및 새로운 종의 기능적 접근 방법의 급속?...

1998 년 화재와 멜로는 이중 가닥 RNA (dsRNA를이) 예쁜 꼬마 선충 1 유전자 기능의 억제를 유도 할 수 있다고보고했다. dsRNA에 의해 유발이 반응은 RNA 간섭 (RNAi의) 명명하고, 이러한 RNAi를 매개 된 유전자 사일런 싱은 동물, 식물, 균류 및 2-7에서 보존되는 것으로보고되었다. 일부 식물 및 동물에서의 RNAi 기능 전신적 의미 효과 (8-10에서 검토) dsRNA를 직접 도입되지 않은 다른 세포 / 조직으로 확산 될 수있다. 과학자하여 직접 (11-14에서 검토) 게놈을 조작없이 유전자 기능을 쓰러 뜨린, 관심의 유전자를 대상으로 dsRNA에를 설계하여이 내생 세포의 RNAi 응답의 사용을 만들었습니다. RNAi의는 다음과 같은 장점으로 인해 기능 연구를위한 강력한 도구입니다. 먼저도 최소한의 유전자 서열 정보하는 유전자의 RNAi를 사용하여 타겟팅 될 수있다. 이 일에 특히 중요하다게놈 또는 transcriptomic 데이터가없는 비 모델 생물의 udies. 둘째, RNAi의 응답이 견고 전신 인 유기체에서 RNAi를 매개하는 유전자 녹다운 거의 모든 발육 단계에서 수행 될 수있다. 이 기능은다면 발현 유전자의 기능을 연구하는데 매우 유용하다. 셋째, 어떤 경우에는, RNAi의 효과 표현형 자손 15,16 관찰되도록 상기 생식선 및 자손에 퍼졌다. 단일 주입 부모에 의해 생성 된 수많은 자손 계란을 직접 조작하지 않고 검사 할 수있는 부모의 RNAi (pRNAi)로 알려진이 현상은,,, 배아 발달에 영향을 미치는 유전자에 특히 유리하다. 이러한 이유로, pRNAi는 선택의 방법입니다. pRNAi 비효율적 인 경우, oogenesis 필요한 유전자, 예를 들어 다음의 RNAi 배아 (eRNAi)이 사용되어야한다. 넷째, RNAi의 강력한 결함 약한 생산하는 dsRNA의 양은 범위에 걸쳐 변화 될 수있는 전달하는 대립 시리즈의 등가를 생성하는데 사용될 수있다. 표현형 그러한 계조 유전자가 치명적인 복잡한 프로세스 및 / 또는 기능을 완전히 상실 포함될 때 유전자 기능을 이해하는데 도움이 될 수있다. 다섯째, dsRNA를 전달 특히 강력한 조직의 RNAi 반응을 나타내는 동물, 일반적으로 쉽게 가능하다. dsRNA를이 미세 주입 1,5, 공급 / 섭취 17, 18, 침지, 19, 20 및 바이러스 / 박테리아 매개 배달 (21, 22)에 의해 도입 될 수있다. 여섯째, 일부 유전자 타겟팅 / 편집 방법과 달리, 돌연변이를 운반 생물을 선별하거나 RNAi의 사용시 동형 접합체를 생성하기 위해 유전 교배를 실시 할 필요가 없다. 따라서, 유전자 기능을 연구하기위한 다양한 다른 기술에 비해 저렴 RNAi를 빠르게하고, 대형 화면 (23-25)에 적용될 수있다. 의 RNAi의 다양한 유틸리티 beyon 연구 가능한 종의 범위를 확대하고, 생물의 광범위한 기능적 연구를 수행하는 방법을 제공한다유전 적 도구가 개발되어있는 기존의 모델 시스템 거라고. 예를 들어, 비 - 모델 시스템을 사용하여 연구가 다른 개발 모드를 표시하거나 별개의 형태 적 특징 26-29을 나타내는 종 orthologs의 기능을 비교하여 유전자 및 유전자 네트워크의 발전에 대한 통찰력을 제공해야한다. 연구 이러한 유형의 응용 및 기본적인 두 연구의 영향이 생물학적 다양성의 더 나은 이해를 제공 할 것이다. 지구상에서 가장 큰 동물 그룹이기 때문에, 곤충 메커니즘을 기본 다양성을 탐험 할 수있는 좋은 기회를 제공합니다. 또한, 곤충은 일반적으로 작고 짧은 수명주기, 높은 생산력을 가지고 있고, 실험실에서 뒤쪽으로 쉽습니다. 지난 20 년에, RNAi의 성공적 파리목 (사실 파리) 5, 나비목 (나비와 나방) (30)를 포함하여 주문에 걸쳐 곤충 적용되어, 딱정벌레 목 (딱정벌레) 16,31, 벌목 (sawf거짓말, 말벌, 개미와 꿀벌) (32), 매미목 (사실 버그), Isoptera (흰개미) (34), Blattodea (바퀴벌레) (35), 메뚜기 (귀뚜라미, 메뚜기, 메뚜기, 그리고 katydids) 36 Phthiraptera (이가) 37. RNAi를 성공적으로 응용 프로그램, 에크 디손 (42) 신호, 초기 배아에서 패턴의 연구 기능 데이터 (전후방 축 (32), 지느러미 - 복부 축 (28), 세그멘테이션 26,38), 성별 결정 39, 40, 키틴 / 표피의 생합성 (41)를 제공하고있다 사회적 행동 (43), 그리고 더. 다른 곤충 종 개발의 RNAi 방법은 (44-46 검토) 방제에 유용 할 가능성이 있다는 점에서 추가적인 이점이 될 수있다. RNAi의 효과는 비 보존 지역을 타겟팅 선택되는만큼, 유전자 특이뿐만 아니라 종 특이 할 것이다. 의 생존을위한 필수적인 유전자를 대상으로 꿀벌과 누에와 같은 유익한 곤충 종에 대한감염을 제어하는 바이러스 또는 기생충이 종 (47, 48)을 보호하기위한 새로운 전략을 제공 할 수있다. Dermestes maculatus (D. maculatus), 일반 이름 숨기기 딱정벌레, 남극 대륙을 제외하고 전 세계적으로 배포됩니다. holometabolous 곤충으로, D. maculatus 수명주기는 배아, 애벌레, 번데기 및 성인 단계 (그림 1)을 포함한다. 그것은 육체에 공급하기 때문에, D. maculatus는 죽은 동물을 해골로하는 박물관으로 사용되며, 법의학 곤충 학자 죽음 49,50의 시간을 예상하는 데 사용할 수 있습니다. D. maculatus는 시체, 말린 고기, 치즈, 그리고 번데기 / 다른 곤충의 고치를 포함한 동물 제품을 공급함으로써 가정의 손상, 저장 음식, 그리고 실크, 치즈, 육류 산업 51,52됩니다. 이 딱정벌레에서의 RNAi를 적용하면 경제에 미치는 영향을 최소화하기 위해 효율적이고 환경 친화적 인 방법을 제공 할 수있다. 우리 연구소는 새로운 m로 D. maculatus을 사용하고있다ODEL 곤충 세그먼트 (53)를 연구한다. 그것은 단기 및 장기 생식 개발 사이의 전환을 공부하고 그것에게 유용한 종을, 중간 - 생식 개발자로 실험실 양육 의무가있을뿐 아니라, D. maculatus는 기초 연구에 대한 관심이다. <img alt="그림 1" src="/files/ftp_upload/54976/54976fig1.jpg" /> 그림 1 : D. maculatus의 수명주기. 나타낸 바와 같이, 서로 다른 삶의 단계에서 D. maculatus의 사진. 성인에 계란에서 라이프 사이클은 낮은 온도에서 세 30 ° C에서 주하지만 더 오래 걸립니다. (A, F) 갓 배치 배아의 길이는 약 1.5 mm, 밝은 노란색 타원형에 흰색입니다. 배아는 ~ 30 ° C에서 55 시간이 걸립니다. (B, C와 G) 애벌레 어두운 색소 줄무늬가 있고 강모로 덮여있다. 애벌레는 1 이상 cm까지 확장 할 수있는 환경과 길이에 따라 여러 령충을 통해 이동합니다. (D, H) </str옹&gt; 젊은 번데기는 황색이다. 30 ° C에서 칠일 - 번데기는 5 ~합니다. 곧 우화 후 (E, I)는, 어두운 색소 침착은 성인 딱정벌레 몸에 나타납니다. 성인은 몇 개월까지 살 수있는 한 여성은 그녀의 일생을 통해 배아의 수백을 배치 할 수 있습니다. <a href="http://ecsource.jove.com/files/ftp_upload/54976/54976fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> 이전에, 우리는 RNAi의는 D. maculatus (53)에 유전자 기능을 쓰러 뜨린에 효과가 있음을 보여 주었다. 다음은 실험실에서 D. maculatus 식민지를 양육 경험은 배아와 부모 모두 RNAi의 설정, 사출, 포스트 분사 관리 및 표현형 분석을위한 단계별 프로토콜과 함께 공유됩니다. 여기에 소개 된 dsRNA를 매개 유전자 최저 및 분석 방법 D. maculatus에 문제를 해결에 대한 자세한 정보를 제공 할뿐만 아니라, FO 잠재적 인 의미가 아니라R 아닌 다른 모델 딱정벌레 / 곤충 종의 RNAi를 적용.

<table border="0" cellpadding="0" cellspacing="0" width="642">
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dermestes maculatus live beetles</td>
			<td style="width:140px;">Our lab or Carolina Biological Supply</td>
			<td style="width:119px;">#144168</td>
			<td style="width:167px;">Our lab strain was verified by COI barcoding; strain variation from Carolina cannot be ruled out</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Wet cat food</td>
			<td style="width:140px;">Fancy Feast</td>
			<td style="width:119px;"></td>
			<td>Chunks of meat with gravy. Can buy at most pet food and grocery stores</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dry dog food</td>
			<td style="width:140px;">Purina Puppy Chow</td>
			<td style="width:119px;"></td>
			<td>Can buy at most pet food and grocery stores</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect cage (size medium, 30.5x19x20.3 cm)</td>
			<td style="width:140px;">Exo Terra</td>
			<td>PT2260</td>
			<td>For colony maintenance. Can use larger cage if needed</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Insect cage (size mini, 17.8x10.2x12.7 cm)</td>
			<td style="width:140px;">Exo Terra</td>
			<td>PT2250</td>
			<td>For embryo collection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">89038-968</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cotton ball</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">22-456-883</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Megascript T7 transcription kit</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">AM1334</td>
			<td>For 40 reactions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pneumatic pump</td>
			<td style="width:140px;">WPI</td>
			<td style="width:119px;">PV830</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Capillary holder</td>
			<td style="width:140px;">WPI</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:140px;">NARISHIGE</td>
			<td style="width:119px;">MN-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Black filter paper (90 mm)</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">28342-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Food coloring (green)</td>
			<td>McCormick</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Borosilicate glass capillary</td>
			<td style="width:140px;">Hilgenberg</td>
			<td style="width:119px;">1406119</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Needle puller (micropipette puller)</td>
			<td style="width:140px;">Sutter Instrument Co.</td>
			<td style="width:119px;">P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microscope glass slide</td>
			<td>WorldWide Life Sciences Division</td>
			<td style="width:119px;">41351157</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sealing film (Parafilm M)</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">13-374-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Model 801 Syringe (10 &#181;l )</td>
			<td style="width:140px;">Hamilton</td>
			<td style="width:119px;">7642-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle (32-gauge)&#160;</td>
			<td style="width:140px;">Hamilton</td>
			<td style="width:119px;">7762-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fixation Solution (Pampel&#39;s)</td>
			<td>BioQuip Products, Inc.</td>
			<td style="width:119px;">1184C</td>
			<td>Toxic, needs to be handled in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep (DUMONT #5)</td>
			<td style="width:140px;">Fine Science Tools</td>
			<td style="width:119px;">11252-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cover slip (24X50 mm, No. 1.5)</td>
			<td style="width:140px;">Globe Scientific</td>
			<td style="width:119px;">1415-15</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Femtotips Microloader pipette tip</td>
			<td style="width:140px;">Fisher</td>
			<td style="width:119px;">E5242956003</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscopy for embryo injection</td>
			<td style="width:140px;">Leica</td>
			<td style="width:119px;">M420</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscopy for larval phenotypic visualization</td>
			<td style="width:140px;">Zeiss</td>
			<td colspan="2">SteREO Discover. V12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DIC microscopy</td>
			<td style="width:140px;">Zeiss</td>
			<td style="width:119px;">AXIO Imager. M1</td>
			<td></td>
		</tr>
	</tbody>
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rearing and double-stranded rna-mediated gene knockdown in the hide beetle, dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

저자의 실험실은 곤충 53, 55의 분할을 조절하는 유전자의 기능 진화를 연구하는 RNAi의 기술을 사용하고있다. 모든 곤충이 분할되어 있지만, 이러한 과정을 조절하는 유전자를 곤충 방사선 26,38,56-63 동안 갈라 것으로 보인다. 초파리 유전자 스크린 바디 세그먼트 64-70의 형성을 촉진하는 책임 아홉 쌍 룰 분할 유전자 세트를 식별. 여기서, 이?...

Watch this Scientific Journal Video about Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus at JoVE.com

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

여기, 우리는 실험실에서 중간 - 세균 딱정벌레, Dermestes maculatus (D. maculatus)를 양육하기위한 프로토콜을 제시한다. 우리는 또한이 종에서 유전자 기능을 연구하기 위해 배아 표현형을 분석 배아 및 부모의 RNAi를위한 프로토콜과 방법을 공유 할 수 있습니다.

양육 및 숨기기 비틀에 RNA 매개 유전자 최저를 두 번 좌초,<em&gt; Dermestes maculatus</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

rearing-double-stranded-rna-mediated-gene-knockdown-hide-beetle

Research

JoVE Journal

Genetics

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

10.5K 조회수.  University of Maryland.  여기, 우리는 실험실에서 중간 - 세균 딱정벌레, Dermestes maculatus (D. maculatus)를 양육하기위한 프로토콜을 제시한다. 우리는 또한이 종에서 유전자 기능을 연구하기 위해 배아 표현형을 분석 배아 및 부모의 RNAi를위한 프로토콜과 방법을 공유 할 수 있습니다. 

비디오: Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

인간의 기본 세포 배양의 일주기 시계 유전자 발현 및 호르몬 분비의 병렬 측정

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

연구

유전학

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

마우스 마음에서 과발현하는 rAAV9의 제조 또는 최저 유전자

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

두 개의 상보적인 세포 동기화 프로토콜에 의한 세포주기 조절 유전자 발굴 연구

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

점 돌연변이 수리 CRISPR/Cas9 및 SsODN 인간 세포에 의해 촉매의 기능으로 현지 변이 검사 하는 표준 방법론

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

시퀀스 및 선택적 인식 하 여 단일 가닥 RNAs에 더블-좌초 RNAs의 화학적 펩 티 드 핵 산 수정

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

효율적인 생산 및 모델 시스템 Danio rerio 에 유전자 녹아웃 식별의 CRISPR/Cas9 생성

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

생산 및 유전자 치료 응용 프로그램에 대 한 잠재의 정화

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

Thromboxane A2 수용 체 유전자의 C924T 다형성을 공부 하는 방법

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

루트 미생물 탐색: 토양, Rhizosphere, 및 루트 Endosphere에서 세균성 지역 사회 데이터 추출

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

효 모 신경 Proteinopathy 모델에서 특성화 히스톤 포스트 번역 상 수정 변경

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...