이 작품은 대상 중심의 R &amp; D, 일본 과학 기술 진흥기구, 일본의 사립 학교에 대한 홍보 및 공제 Corporation의 과학 연구 진흥 기금을 통해 적응력과 원활한 기술 이전 프로그램에서 지원하고, JSPS KAKENHI ​​그랜트 번호 24300342과 15H04313했다 .

헵 타머 형 1. 건설 sgRNA 도서관 <ol><li> 본격적인 라이브러리의 구축에 사용되지 않는 한, 384 (4-7) -7- NT 시퀀스의 서브 세트를 선택한다. 참고 : 순서는 랜덤 및 / 또는 특정 세포의 RNA를 대상으로 설계 할 수있다. 후자의 경우, 헤어핀 구조 -4,10-의 바로 하류 -7- NT 서열에 상보 적절한 컴퓨터 프로그램 및 / 또는 시각, 선택 시퀀스들을 이용하여 표적 RNA에서의 tRNA의 T 아암 닮은 헤어핀 구조를 찾아 17, 18. </li><li> 로 선택한 heptamers의 각을 합성 완전히 2&#39;- O -methylated, 5&#39;- 및 DNA / RNA 합성과 3&#39;- 인산화 RNA. 제어 된 기공 유리 (CPG) 지지체 상 포스 방법을 사용하여 5&#39;- 말단 4,4&#39;- 디메 톡시 트리 틸 (DMT) 합성의 마지막 사이클에서 (5 &#39;포스페이트를 추가) 보호기를 남겨 23. 참고 : 사용자 정의 한건데nthetic RNA는 뉴클레오티드 공급 업체에서 얻을 수 있습니다. </li><li> 마지막 합성주기의 완료 후, 8 시간 동안 55 ° C에서이를 1 ㎖의 29 % 암모늄 하이드 록 사이드를 추가 테플론 라이너 스크류 캡이있는 유리 병에 컬럼으로부터 합성 올리고 뉴클레오티드와 CPG를 전송하고, 부화 소비재에서 올리고 뉴클레오티드를 절단하고 기지와 인산염에서 보호 그룹을 제거합니다. </li><li> 40 ° C에서 원심 증발기로 암모늄 히드 록 시드를 증발시키고, 0.2 mL의 0.1 M N-hexylammonium 아세테이트로 올리고 뉴클레오티드를 재용. </li><li> 10~50% 아세토 니트릴 / 0.1 M N-hexylammonium 아세테이트를 함유하는 완충액, 폴리스티렌 - 디 비닐 벤젠 칼럼을 사용하는 역상 고성능 액체 크로마토 그래피를 통해 정제하여 합성 올리고 뉴클레오티드. 80 ° C에서 25 분 동안 / 2.0 ml의에서 분 열을 실행합니다. </li><li> 20 % 아세트산 용액을 제조하기 위해 분류 된 샘플 (1-2 ㎖)로 농축 아세트산 0.25-0.5 mL를 넣고 incubaTE, 실온에서 1 시간 동안이 용액은 DMT기를 제거한다. </li><li> 40 ° C에서 원심 증발기를 사용하여 샘​​플을 건조하고 1 ml의 29 % 암모늄 하이드 록 사이드에서 그것을 재 - 용해하고, 5 &#39;포스페이트의 측쇄를 제거하여 실온에서 15 분 동안 용액을 배양한다. </li><li> 40 ° C에서 원심 증발기로 약 0.3 ml의 시료의 부피 (1 ml)로 줄인다. </li><li> 차단 1000을 4 ℃에서 밤새 분자량 투석 튜브를 사용하여 증류수에 대한 전체 농축 샘플 (500 ml)에 투석. </li><li> 그 후, 피펫으로 1.5 ㎖의 튜브로 투석 튜브로 투석 샘플을 이동시켜 40 ℃에서 원심 증발기로 건조하고, 볼륨있는 경우, 물에 대한 주입 등급 물에 재용 더 큰 100 μM 솔루션을 만들기에 충분 작은. </li><li> 분광 광도계로 260 nm에서의 RNA 시료의 광학 밀도를 측정하고 (100)의 농도를 조정물에 대한 주입 등급의 물을 첨가하여 μM. -20 ° C 사용하기 전에 아래의 RNA 솔루션을 저장합니다. </li></ol> 세포 생존에 대한 sgRNA 도서관 2. 심사 <ol><li> 준비 103 세포 / 99 μL / 웰 (예 : HL60 및 RPMI-8226) 10 % 소 태아 혈청과 1 % 페니실린 - 스트렙토 마이신 용액이 보충 된 RPMI-1640 배지에서 96 웰 접시. 단지 배경 공제 미디어를 포함하는 우물을 준비합니다. 샘플 우물 주변 우물을 비우 멸균 물을 추가, 가장자리 효과를 제거합니다. </li><li> 각 웰에 물에 대한 주입 수준의 물에 용해 헵 타머 형 sgRNA (100 μM) 1 μl를 추가합니다. 세중 각 sgRNA를 분석. 3 일간 5 % CO 2 가습 배양기에서 37 ℃에서 플레이트를 인큐베이션. </li><li> 각 웰에 시판 WST-8 솔루션의 적절한 볼륨을 추가합니다. 1 내지 4 시간 동안 동일한 인큐베이터에서 37 ℃에서 플레이트를 인큐베이션. </li><li> 흡광도 A를 측정마이크로 플레이트 리더 450 nm의에서 t. 주 : 대부분의 경우, 흡광도 및 생존 세포 수 사이의 선형성 흡광도 값이 1.0를 초과하지 않는 한 유지 될 것으로 보인다. </li></ol> 유동 세포 계측법에 의한 효과적인 sgRNAs 3. 평가 <ol><li> 준비 103 세포 / 10 % 소 태아 혈청과 1 % 페니실린 - 스트렙토 마이신 용액이 보충 된 RPMI-1640 배지에서 96 웰 접시에 99 μL / 웰 (예 : HL60). 하나 sgRNA가 테스트 될 때까지 적어도 10 우물을 준비합니다. 샘플 우물 주변 우물을 비우 멸균 물을 추가, 가장자리 효과를 제거합니다. </li><li> 각 웰에 물에 대한 주입 수준의 물에 용해 헵 타머 형 sgRNA (100 μM) 1 μl를 추가합니다. 3 일간 5 % CO 2 가습 배양기에서 37 ℃에서 플레이트를 인큐베이션. </li><li> 피펫으로 5 ㎖의 튜브에 적어도 10 웰로부터 동일한 sgRNA로 처리 된 세포를 수집한다. 30 원심 분리기에서 5 분 동안 튜브를 스핀0 XG, 미디어를 폐기합니다. </li><li> 튜브로 1X 인산 완충 생리 식염수 (PBS) 2 mL를 넣고 세포를 다시 일시. 300 XG에 원심 분리기에서 5 분 동안 튜브를 스핀, 그리고 PBS를 폐기합니다. 튜브에 1 배 넥신 V 결합 버퍼의 150 μl를 추가하고 세포를 다시 일시 중지합니다. </li><li> , 피코 에리 트린의 2 μL (PE) π 공역 넥신 V 용액 튜브 내지 7 aminoactinomycin의 D (7AAD) 액 1 μl를 추가들을 잘 혼합하고, 어둠에서 실온에서 15 분 동안 튜브를 배양한다. (24) 플로우 사이토 미터를 사용하여 샘플을 분석한다. </li></ol>

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Niigata University of Pharmacy and Applied Life Sciences has a pending patent application on a subset of the heptamer-type sgRNAs.

In most cases, fully 2&#8242;-O-methylated, 5&#8242;- and 3&#8242;-phosphorylated heptamer-type sgRNAs dissolved in water-for-injection-grade water (in the concentration of 100 &#181;M) appear to be stable at below -20 &#176;C for at least one year, judging from their activity to induce apoptosis in cancer cells. However, their stability would change depending on their sequence, quality, and purity. In some cases, 5&#8242;- or 3&#8242;-phosphate of a part of sgRNA molecules appears to be removed spontaneously du...

(TR Nase Z의 L 후 라 - 유 전자 fficacious 유전자 침묵을 tilizing) TRUE 유전자 침묵은 RNA 지시 유전자 침묵 기술 (1) 중 하나입니다. 이 기술은 개발 tRNase Z L (또는 3 &#39;tRNase), tRNA의 3&#39;처리 endoribonuclease의 속성을 기반으로하고있다 : 그것은을 인식하여 인공 작은 가이드 RNA (sgRNA)의 지시에 따라 어떤 예상 사이트에있는 모든 대상 RNA를 절단 할 수 있습니다 9 - 표적 RNA와 sgRNA (2) 사이에 형성된 프리 tRNA의 형상 또는 마이크로 사전의 tRNA 형 구조. 길이 NT를 일반적으로 7-31이다 sgRNA는 네 그룹, 5&#39;- 절반의 tRNA, 헵 타머 RNA, 14 NT를 선형 RNA, 그리고 훅 RNA로 분류된다. 15 - 우리는 셀 (10) 생활에 다양한 인위적으로 설계 sgRNAs을 도입하여 TRUE 유전자 침묵의 효과를 증명하고있다. 우리는 또한 sgRNAs이 세포 위스콘신에 의해 흡수 될 수 있음을 보여 주었다모든 형질 전환 시약 사전 통 보없이 그들의 표적 RNA 수준을 하향 조절 및 / 또는 인간 암세포 (16)에 세포 자멸을 유도 할 수있다 - 18. 21 - TRUE 유전자 침묵은 tRNase Z L과 자연 sgRNAs (19)를 통해 세포 내와 세포 간 유전자 조절 네트워크 시스템에서 작동하도록 나타납니다. 우리는 혈액 암 (22)에 대한 잠재적 인 치료 헵 타머 형 sgRNAs 검색 156 헵 타머 형 sgRNAs 함유 sgRNA 라이브러리를 구축했다. 우리는 인간 골수종 및 백혈병 세포의 생존에 대한 효과를 스크리닝하고 20 효율적으로 암세포의 적어도 하나의 세포 사멸을 유도 할 수 있음을 발견 하였다. 그 중 4 마우스 이종 이식 실험에서 종양 성장 속도를 감소시키는 것으로 나타났다. 여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다. 이 프로토콜은 어떻게 포함세포 생존, 어떻게 더 유동 세포 계측법에 의해 유효 sgRNAs을 평가하는 각 sgRNA의 효과를 평가하는 방법 sgRNA 라이브러리를 구성합니다. 이러한 프로토콜이 포함되지 않더라도 마우스 이종 이식 모델에서의 셀룰러 sgRNA 표적 RNA를위한 분석 및 sgRNAs 효과의 평가는, 17, 22 종래의 방법에 따라 수행 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" width="948">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Custom heptamer RNA</td>
			<td style="width:167px;">Nippon Bioservice</td>
			<td style="width:144px;"></td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OligoSep Prep HC Cartridge</td>
			<td style="width:167px;">Transgenomic</td>
			<td style="width:144px;">99-3860</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Water-for-injection-grade Water</td>
			<td style="width:167px;">Otsuka Pharmaceutical&#160;</td>
			<td style="width:144px;">7131400A2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI-1640 medium</td>
			<td style="width:167px;">Wako</td>
			<td style="width:144px;">189-02025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">172012-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:457px;">Penicillin-Streptomycin Mixed Solution</td>
			<td style="width:167px;">Nacalai Tesque</td>
			<td style="width:144px;">26253-84</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 Well Plate</td>
			<td style="width:167px;">Greiner Bio-one</td>
			<td style="width:144px;">655 180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Counting Kit-8</td>
			<td style="width:167px;">DOJINDO</td>
			<td style="width:144px;">343-07623</td>
			<td>WST-8 solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate Reader, Sunrise Thermo RC-R</td>
			<td style="width:167px;">TEKAN</td>
			<td style="width:144px;">510-82851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FACS Round-Bottom Tube</td>
			<td style="width:167px;">BD Falcon</td>
			<td style="width:144px;">60819-820</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">P7059-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V Binding Buffer</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-66121E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE Annexin V</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">51-65875X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7AAD</td>
			<td style="width:167px;">SIGMA-ALDRICH</td>
			<td style="width:144px;">A9400-1MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flow Cytometer, FACSCalibur</td>
			<td style="width:167px;">BD Biosciences</td>
			<td style="width:144px;">342973</td>
			<td></td>
		</tr>
	</tbody>
</table>

true gene silencing: screening of a heptamer-type small guide rna library for potential cancer therapeutic agents

TRUE 유전자 침묵은 (TR Nase Z의 L 후 라 - 유 전자 fficacious 유전자 침묵 tilizing)의 tRNA 3 &#39;처리 endoribonuclease, tRNase Z L을 안내하는 인공 작은 가이드 RNA (sgRNA)을 이용 기술을 입을 RNA의 지시 유전자 중 하나입니다 , 표적 RNA를 인식한다. sgRNAs 모든 형질 전환 시약없이 세포에 의해 용해 될 수 있고, 그들의 표적 RNA 수준을 하향 조절 및 / 또는 인간의 암세포에서 아폽토시스를 유도 할 수있다. 우리는 인간 골수종 및 백혈병 세포의 생존에 미치는 영향 156 헵 타머 형 sgRNAs 함유 sgRNA 라이브러리를 스크리닝하고 20 효율적으로 암세포의 적어도 하나의 세포 사멸을 유도 할 수 있음을 발견 하였다. 여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다. 이 프로토콜은 세포 생존, 그리고 각 sgRNA의 효과를 평가하는 방법 쿵푸를하는 방법 sgRNA 라이브러리를 구성하는 방법을 포함rther는 유동 세포 계측법에 의해 유효 sgRNAs을 평가합니다. 약 2,000 명중은 16,384 heptamers 이루어지는 본격적인 sgRNA 라이브러리를 스크리닝함으로써 얻어 질 것으로 예상된다.

여섯 헵 타머 형 sgRNAs 5&#39;- AUCUUCA-3 &#39;(H1885), 5&#39;- ACACACA-3&#39;(H3277), 5&#39;- GGGGGCG-3 &#39;(H10927), 5&#39;- GGGGCCC-3&#39;(H10944), 5&#39;-GCCCCCG-3 &#39;(H12287), 및 5&#39;-CACCAGC-3&#39;(H13260)을 화학적으로 3&#39;- 및 5&#39;- 포스페이트를 함유하는 O -2&#39;- 메틸 RNA 합성 하였다. 이러한 sgRNAs은 인간 백혈병 세포주, HL60과 인간 골수종 세포주, RPM...

Watch this Scientific Journal Video about TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents at JoVE.com

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

여기에서 우리는 혈액 암에 대한 잠재적 인 치료 약물에 대한 헵 타머 형 sgRNA 라이브러리의 스크리닝을위한 프로토콜을 제시한다.

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 ...

The overall goal of this screening is to find potential therapeutic drugs against blood cancers.Begin this procedure by selecting a subset of Heptamer sequences.The sequences can be random, or designed to target specific cellular RNAs.To target specific cellular RNAs, use an appropriate computer program to find hairpin structures resembling the T-arm in a target RNA.Select complementary seven-nucleotide sequences immediately downstream of the hairpin.Using the phosphoramidite method on the controlled pore glass, or CPG support, synthesize each of the selected Heptamers as a fully 2-prime O-methylated, 5-prime and 3-prime phosphorylated RNA with the DNA-RNA synthesizer.Leave the 5-prime terminal DMT protecting group on in the last cycle of the synthesis.After completion of the last cycle, transfer the CPG with the synthesized oligonucleotide from the column to a glass vial with a teflon-lined screw cap.Add 1mL of 29%ammonium hydroxide and incubate the tube at 55

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5.6K 조회수.  Niigata University of Pharmacy and Applied Life Sciences. The overall goal of this screening is to find potential therapeutic drugs against blood cancers.Begin this procedure by selecting a subset of Heptamer sequences.The sequences can be random, or designed to target specific cellular RNAs.To target specific cellular RNAs, use an appropriate computer program to find hairpin structures resembling the T-arm in a target RNA.Select complementary seven-nucleotide sequences immediately downstream of the hairpin.Using the phosphoramidite method on the co...

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

Bronchial Thermoplasty:  A Novel Therapeutic Approach to Severe Asthma

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks. Bronchial thermoplasty is delivered by the Alair System and is performed in three outpatient procedure visits, each scheduled approximately three weeks apart. The first procedure treats the airways of the right lower lobe, the second treats the airways of the left lower lobe and the third and final procedure treats the airways in both upper lobes. After all three procedures are performed the bronchial thermoplasty treatment is complete.
Bronchial thermoplasty is performed during bronchoscopy with the patient under moderate sedation. All accessible airways distal to the mainstem bronchi between 3 and 10 mm in diameter, with the exception of the right middle lobe, are treated under bronchoscopic visualization. Contiguous and non-overlapping activations of the device are used, moving from distal to proximal along the length of the airway, and systematically from airway to airway as described previously. Although conceptually straightforward, the actual execution of bronchial thermoplasty is quite intricate and procedural duration for the treatment of a single lobe is often substantially longer than encountered during routine bronchoscopy. As such, bronchial thermoplasty should be considered a complex interventional bronchoscopy and is intended for the experienced bronchoscopist. Optimal patient management is critical in any such complex and longer duration bronchoscopic procedure. This article discusses the importance of careful patient selection, patient preparation, patient management, procedure duration, postoperative care and follow-up to ensure that bronchial thermoplasty is performed safely.
Bronchial thermoplasty is expected to complement asthma maintenance medications by providing long-lasting asthma control and improving asthma-related quality of life of patients with severe asthma. In addition, bronchial thermoplasty has been demonstrated to reduce severe exacerbations (asthma attacks) emergency rooms visits for respiratory symptoms, and time lost from work, school and other daily activities due to asthma.

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled manner to reduce excessive airway smooth muscle. Reducing airway smooth muscle decreases the ability of the airways to constrict, thereby reducing the frequency of asthma attacks.

기관지 Thermoplasty : 심한 천식에 소설 치료 접근

Bronchial thermoplasty is a non-drug procedure for severe persistent asthma that delivers thermal energy to the airway wall in a precisely controlled ...

연구

의학

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to inhibit tumor growth must be performed in a suitable animal model. The type of animal model that is best for this purpose is a topic of intense discussion. Some evidence indicates that preclinical trials examining drug effects on tumors arising in transgenic mice are more predictive of clinical outcome1and so candidate therapeutic agents are often tested in these models. Unfortunately, transgenic models are not available for many tumor types. Further, transgenic models often have other limitations such as concerns as to how well the mouse tumor models its human counterpart, incomplete penetrance of the tumor phenotype and an inability to predict when tumors will develop.
Consequently, many investigators use xenograft models (human tumor cells grafted into immunodeficient mice) for preclinical trials if appropriate transgenic tumor models are not available. Even if transgenic models are available, they are often partnered with xenograft models as the latter facilitate rapid determination of therapeutic ranges. Further, this partnership allows a comparison of the effectiveness of the agent in transgenic tumors and genuine human tumor cells. Historically, xenografting has often been performed by injecting tumor cells subcutaneously (ectopic xenografts). This technique is rapid and reproducible, relatively inexpensive and allows continuous quantitation of tumor growth during the therapeutic period2. However, the subcutaneous space is not the normal microenvironment for most neoplasms and so results obtained with ectopic xenografting can be misleading due to factors such as an absence of organ-specific expression of host tissue and tumor genes. It has thus been strongly recommended that ectopic grafting studies be replaced or complemented by studies in which human tumor cells are grafted into their tissue of origin (orthotopic xenografting)2. Unfortunately, implementation of this recommendation is often thwarted by the fact that orthotopic xenografting methodologies have not yet been developed for many tumor types.
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive sarcomas that occur sporadically or in association with neurofibromatosis type 13and most commonly arise in the sciatic nerve4. Here we describe a technically straightforward method in which firefly luciferase-tagged human MPNST cells are orthopically xenografted into the sciatic nerve of immunodeficient mice. Our approach to assessing the success of the grafting procedure in individual animals and subsequent non-biased randomization into study groups is also discussed.

Orthotopic Xenografting of Human Luciferase-Tagged Malignant Peripheral Nerve Sheath Tumor Cells for in vivo Testing of Candidate Therapeutic Agents

A method for reliably grafting luciferase-tagged human malignant peripheral nerve sheath tumor cells into the sciatic nerve of immunodeficient mice is described. The use of bioluminescence imaging to demonstrate proper establishment of tumor grafts and criteria for random segregation of animals into study groups are also discussed.

대한 인간 루시페라제 태그가 추가된 악성 주변 신경 쉬스 종양 세포 Orthotopic Xenografting 생체내에</em후보 치료 대리점> 테스트

Although in vitro screens are essential for the initial identification of candidate therapeutic agents, a rigorous assessment of the drug's ability to ...

Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most often results in failure of potential therapeutics designed for neurodegenerative disorders 1,2. Neurodegenerative diseases such as Spinal Muscular Atrophy (SMA), in which the lower motor neurons are affected, can benefit greatly from introducing the therapeutic agents into the CNS. The purpose of this video is to demonstrate two different injection paradigms to deliver therapeutic materials into neonatal mice soon after birth. One of these methods is injecting directly into cerebral lateral ventricles (Intracerebroventricular) which results in delivery of materials into the CNS through the cerebrospinal fluid 3,4. The second method is a temporal vein injection (intravenous) that can introduce different therapeutics into the circulatory system, leading to systemic delivery including the CNS 5. Widespread transduction of the CNS is achievable if an appropriate viral vector and viral serotype is utilized. Visualization and utilization of the temporal vein for injection is feasible up to postnatal day 6. However, if the delivered material is intended to reach the CNS, these injections should take place while the blood brain barrier is more permeable due to its immature status, preferably prior to postnatal day 2. The fully developed blood brain barrier greatly limits the effectiveness of intravenous delivery. Both delivery systems are simple and effective once the surgical aptitude is achieved. They do not require any extensive surgical devices and can be performed by a single person. However, these techniques are not without challenges. The small size of postnatal day 2 pups and the subsequent small target areas can make the injections difficult to perform and initially challenging to replicate.

Delivery of Therapeutic Agents Through Intracerebroventricular ICV and Intravenous IV Injection in Mice

This article demonstrates two very different methods of injection: 1) into the brain (intracerebroventricular) and 2) systemic (intravenous) to introduce the therapeutic agents into the central nervous system of neonatal mice.

마우스에 Intracerebroventricular을 통해 치료 대리점 납품 (ICV)과 정맥 (IV) 사출

Despite the protective role that blood brain barrier plays in shielding the brain, it limits the access to the central nervous system (CNS) which most ...

A Simple Guide Screw Method for Intracranial Xenograft Studies in Mice

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma (glioma) and medulloblastoma. The widely used stereotactic approach only allows for the injection of a single animal at a time, is labor intensive and requires highly specialized equipment. The guide screw method, initially developed by Lal et al.,1 was developed to eliminate cumbersome stereotactic procedures. We now describe a modified guide screw approach that is rapid and exceptionally safe; both of which are critical ethical considerations. Notably, our procedure now incorporates an infusion pump that allows up to 10 animals to be simultaneously injected with tumor cells.
To demonstrate the utility of this procedure, we established human U87MG glioma cells as intracranial xenografts in mice, which were then treated with AMG102; a fully human antibody directed to HGF/scatter factor currently undergoing clinical evaluation2-5. Systemic injection of AMG102 significantly prolonged the survival of all mice with intracranial U87MG xenografts and resulted in a number of complete cures. 
This study demonstrates that the guide screw method is an inexpensive, highly reproducible approach for establishing intracranial xenografts. Furthermore, it provides a relevant physiological model for validating novel therapeutic strategies for the treatment of brain cancers.

In order to evaluate novel therapeutic paradigms for the treatment of glioma, physiological relevant models are essential. We utilize an implantable guide screw procedure for establishment of intracranial xenograft models that is more rapid and safer than stereotactic approaches.

마우스에 Intracranial 이종 이식 연구에 대한 간단한 안내 스크류 방법

The grafting of human tumor cells into the brain of immunosuppressed mice is an established method for the study of brain cancers including glioblastoma ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

암 관련 유전자 기능을 연구하기 DNA를 벡터 기반의 RNA 간섭

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

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 ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Zebrafish Autochthonous 종양 모델을 사용하여 흑종 조절을위한 검사

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

유전자 전달 연구 동소 방광암 모델

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

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.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

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 ...

Disposable Dosators for Pulmonary Insufflation of Therapeutic Agents to Small Animals

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver drugs locally and systemically. Evaluation of dry powder aerosols necessitates an efficient dispersion mechanism to maintain high concentrations in an exposure chamber or for direct endotracheal administration. While solutions exist to expose animals by passive inhalation to dry powder aerosols, most require masses of powder in large excess of the dose delivered. This precludes conducting early feasibility studies as insufficient drug is available at the research or early development stage to support the dose delivery requirements for conventional aerosol delivery systems. When designing an aerosol drug product, aerodynamic performance can relate directly to delivery efficiency and efficacy. Dispersion of powder into an aerosol requires energy input sufficient to overcome interparticulate forces, and particle engineering approaches can substantially improve aerosol performance. We have developed a dispersion system (dosator) which can aerosolize engineered dry powder aerosols efficiently for the purpose of direct pulmonary insufflation, dispersion into an exposure system or generation for analytical purposes.

During development of drugs for pulmonary delivery, it is necessary to evaluate pharmacokinetics and efficacy in an animal model. We present a method to build a disposable aerosol dispersion system from of-the-shelf components that can be used to administer intrapulmonary dry powder aerosol to rodents.

작은 동물에 대한 치료제의 폐 통기를위한 일회용 Dosators

Development of new therapeutic products requires efficacy testing in an animal model. The pulmonary route of administration can be utilized to deliver ...