Work related to this technique is supported by NIH/NICHD RO1 HD070044 and USDA/NIFA Hatch projects W-3171 and W-2112.

1. 마이크로 피펫 생산 <ol><li> 사출 마이크로 피펫 <ol><li> 보로 실리케이트 유리 모세관 (1.0 mm, 내경 (ID) : 외경 (OD) 0.75 mm) 배치 마이크로 피펫 풀러에서 (오른쪽과 왼쪽 모세관 홀더의 중심을)하고 잠급니다. </li><li> 그것은 긴 캐처 얇은 팁을 초래하므로 유리 모세관을 당겨 적절한 프로그램을 사용하십시오. (예 : 열 : 825; 당겨 : 30; 속도 : 120; 시간 : 200; 압력 : 500). </li><li> 조심스럽게 장치에서 뽑아 피펫을 제거하고 수평 위치에 microforge에 놓습니다. </li><li> 인출 된 피펫과 초점에 그 유리 구슬과 microforge 히터 필라멘트를 가져옵니다. 원하는 두께를 측정하고 히터 필라멘트 대상 내경 (5 μm의)에 도달 할 때까지 수평으로 피펫을 이동 접안 레티클을 사용합니다. </li><li> 약 45 %의 열을 조절하고 필라멘트에 닿을 수 있도록 부드럽게 아래로 피펫을 가지고. 잠시 히터를 활성화합니다. 이 w병 약간 필라멘트에 접착되도록 피펫을 용융 및 냉각시에, 피펫 직선 절단을 생성하는 접촉 지점에서 중단된다. 참고 : 너무 높은 온도가 피펫 벤드를 만들 것입니다 따라서 직선 절단 할 수 없습니다 너무 낮은 온도가 피펫 (그림 1A)를 용융하기에 충분하지 않을 것이기 때문에 적절한 온도를 설정하면이 단계의 핵심이다. </li><li> 히터 필라멘트에서 약 10 μm의 멀리 위치하여 피펫 팁 근처 대략 30 ° 각도를 확인합니다. 60 %의 온도를 상기 히터를 작동. 참고 :이 필라멘트를 통해 피펫을 구부 것입니다. 이 각도는 인젝터 (도 1b)에 장착 될 때 바늘의 선단이 주입 판 표면에 평행하도록 요구된다. </li><li> 그들은 매우 선명하고 깨지기 쉬운만큼주의를 미세 주입 피펫을 처리합니다. </li></ol></li><li> 개최 마이크로 피펫 <ol><li> borosilic 배치(: 1.0 mm, ID : OD 0.75 mm) 유리 모세관 먹었다 (오른쪽과 왼쪽 모세관 홀더의 중앙에) 마이크로 피펫 풀러의를하고 잠급니다. </li><li> 이 긴 경우에도 테이퍼 및 병렬 벽 얇은 팁을 초래하므로 유리 모세관을 당겨 적절한 프로그램을 사용하여 (: 열 : 예 815; 당겨 : 20; 속도를 : 140; 시간 : 175; 압력 : 200). </li><li> 조심스럽게 끌어 당기는 장치에서 뽑아 피펫을 제거하고 수평 위치에 microforge 홀더에 놓습니다. 히터 필라멘트 및 피펫에 초점을 조정합니다. </li><li> 피펫 직경을 측정하고 필라멘트를 통해 직경 180 μm의에 도달 할 때까지 피펫을 이동 접안 레티클을 사용합니다. </li><li> 지정된 크기에서, 다이아몬드 팁 펜으로 유리 모세관을 표시 한 다음 부드럽게 유리를 깰 수있는 뽑아 끝에 누릅니다. 이 직선 절단 (그림 1C)가 발생한다. </li><li> 필라멘트에 가까운 그것의 끝이 수직 위치로 피펫을 이동합니다. 온도를 설정<html> 60 %에 microforge의. </li><li> 화재 폴란드어는 40 μm의의 ID에 도달 할 때까지 표준 기술 7을 사용 피펫의 팁. 아이디 (그림 1D)를 확인하기 위해 접안 레티클을 사용합니다. </li><li> 피펫에 각도를 확인합니다. <ol><li> 약 10 μm의 거리에 필라멘트와 거리의 끝에서 5mm에서 다시 수평 위치로 피펫을 가져옵니다. 약 60 %로 온도를 설정하고 필라멘트를 통해 피펫이 구부러 히터를 활성화합니다. (30 O의) 소정의 각도에 도달 할 때까지 가열을 계속한다. 각도 시각 (그림 1E)에서 확인하십시오. 주 : 피펫은 일반적으로 사출 접시의 바닥에 대해 60 (O)의 대략적인 각도에서 마이크로 인젝터에 장착된다. 그것의 중간 평면에서 배아의 정확한 주입에 필요한 접시의 바닥에 평행하도록 피펫의 선단은 절곡되어있다. </li></ol></li></ol></li></ol>제작 &quot;&gt; 2. 미세 조작기 설치 <ol><li> microinjectors가 완전히 기름을 적재되는 시스템에 기포가 없는지합니다 (마이크로 인젝터에 기포가 주입의 미세 제어를 방지) 확인합니다. </li><li> 미세 조작기는 중앙 위치에 있는지 확인하십시오. 이는 피펫의 이동의 넓은 범위를 허용한다. </li><li> 왼쪽 micromanipulator에 홀더에 들고 피펫 오른쪽 미세 조작 홀더에 주입 피펫을 삽입합니다. </li><li> 오일이 모세관 현상에 의해 피펫에 입력하고 시스템이 마이크로 인젝터 컨트롤을 사용하여 피펫 내부 위아래로 오일을 이동하여 제대로 작동하는지 확인할 수 있습니다. 주 : 바늘의 내부에는 오일의 움직임이없는 경우, 막힘이있을 수도있다. 이 경우, 새로운 니들을 사용한다. </li><li> 보기의 현미경의 필드의 중심에 피펫을 가지고하는 미세 조작기 컨트롤을 사용합니다. 4 배의 배율을 사용하여 마이크로 피펫 팁 (올바른 각도로 페이지 있는지 확인접시의 바닥)에 arallel. 참고 : 바늘의 올바른 설치가 성공적으로 주입 및 결과의 일관성을 위해 중요하다. </li><li> 제조자의 설명서 교정 다음 레이저 시스템 교정. </li></ol> 사출 접시 3. 준비 (그림 2) <ol><li> 100mm의 페트리 접시 뚜껑의 중앙에 20 % 소 태아 혈청 (FBS)으로 보충 가온 (37 ° C) SOF-HEPES (재료 부분에 자세히 조성물)의 50 μL를 떨어 놓는다. 용액 2 μL 드롭 주입 드롭에 가까운 주입 할 - 1이 놓습니다. 배아는 RT 이하로 냉각하지 않았는지 확인합니다. 참고 :이 프로토콜에서 우리는 주사 부위를 시각화하고 나중에 추적 할 수 있도록 주입 용액에 덱스 트란 - 붉은 염료를 추가합니다. </li><li> 미네랄 오일을 약 10 ㎖을 사용하여 사출 접시 방울 커버. </li><li> 거꾸로 현미경의 무대에 주입 접시를 놓고 주사위를 가지고분사 방울로 ettes. 바늘 드롭 내부에 초점이 있는지 확인합니다. micromanipulator에 컨트롤을 사용하여 필요에 따라 자신의 높이를 조정합니다. </li><li> 사출 강하의 상측 - (20 시간 후 수정 (HPF) (17)) 30 접합자 - 마이크로 디스펜서를 사용하여 약 20로드. 사출 강하 배아의 개수가 사람을 주입 할 수있는 속도에 의해 결정되도록 상온에서 주입을 수행한다. 30 분에 주입 할 수있는 것보다 더 많은 배아를 넣지 마십시오. <ol><li> 마이크로 디스펜서에 배아를로드하려면 완전히 플런저를 우울하게하고 배아를 포함하는 용액에 끝을 담가. 터치 배아 유리 모세관의 선단 (시 하나)로 촬상 천천히 플런저 각각의 배아 모세관 체결 해제된다. 모든 배아가 선택됩니다 또는 마이크로 디스펜서의 용량이 가득 찰 때까지이 과정을 반복합니다. </li><li> ,로드 된 배아를 해제 전체 볼륨의 공동 때까지 부드럽게 플런저를 우울하게하려면배아를 ntaining하는 모세관에서 해제된다. </li></ol></li></ol> 4. 미세 주입 <ol><li> 4 배의 대물 렌즈를 사용하여 부압 (흡인)을인가하여 주입 피펫으로 주입 할 수있는 용액을로드. 3 접합자 - 2를 주입 할 수있는 충분한 솔루션을로드합니다. 그런 다음 주입 드롭으로 이동합니다. </li><li> 끝이 접합체에 가까운 얻을 수 있도록 주입 드롭 내에서 유지 피펫을 이동합니다. 수정란은 20 배 목표로 유지 피펫과 변화에 고정되는 있도록 유지 마이크로 인젝터와 부압 (흡인)을 적용합니다. </li><li> 수정란이 유지 피펫에 부착되면, 조심스럽게 분리하지 않고 주위의 배아를 이동 주입 피펫을 사용하여 품질을 확인합니다. 좋은 품질의 접합자는 두 극성 몸이 (하나만 볼 가끔 있지만)과 균일 한 세포질한다. 이상 접합자 (그림 3A)를 주입하지 마십시오. </li><li> 필요한 경우, 적절한 주입 배아 재배치위치. 좋은 위치 결정되는 ZP와 PM 사이에 약간의 공간이 때문에 PM이 레이저에 의해 손상되지 않는다는 것이다. </li><li> 주입 피펫은 부드럽게 주사 부위의 접합자를 터치하여 배아의 중간면에 위치되어 있는지 확인합니다. 그것의 중간면에없는 경우, 바늘 배아 대신 천자 회전하게하는 경향이있다. 필요에 따라 주입 피펫의 높이를 조정한다. </li><li> 레이저 (그림 3B)를 사용하여 ZP에 구멍을 만듭니다. <ol><li> 레이저의 소프트웨어 놓고 구멍 (배아가 유지 피펫에 부착되는 곳의 반대측에) 될 것이다 ZP되는 레이저 레티클 사용. 화재 버튼 레이저 제어 창 클릭을 사용. 구멍의 크기가 PM에 손상을주지 않고 통과 바늘 용 ZP에 개구를 생성하기에 충분히 큰지 확인 (예 : 0.662 msec의 펄스 폭 / 6.9 ㎛의 구멍 크기). </li></ol></li><li> 주사 바늘을 통과상기 ZP 구멍을 통해 PM과의 접촉을 만든다. 바늘이 배아 (그림 3C)에 방법의 ¾ 때까지 앞으로 피펫을 눌러 계속합니다. </li><li> 이것이 일관 주입량을 제어 할 수있는 바와 같이, 솔루션 오일 계면에서의 메 니스 커스의 위치를 ​​관찰한다. </li><li> PM과 세포질이 주사 바늘로 흡입 할 것을 발생합니다 주입 피펫에 부압 (흡인)을 적용합니다. 바늘로 세포질의 움직임이 가속 될 때까지 또는 주사 용액과 함께 혼합 세포질 함량을 알 수있는 경우에 계속. 다음은 PM (그림 3D)의 파손을 나타냅니다. </li><li> 용액 유 계면에서의 메 니스 커스가 시작점 과거 1 접합체의 직경에 상당하는 고급까지 양압 (주입)을인가하여 접합체의 세포질 내로 용액 다음 세포질 콘텐츠를 주입한다. 참고 : 우리의 조건이 파트 :변위량 주입 솔루션 (그림 3E)의 약 7 와줘. </li><li> 4 배 목표로 변경하고 주입 드롭의 하부에 주입 된 배아 / s로 이동합니다. 유지 마이크로 인젝터에 양압을 적용하여 배아를 놓습니다. 분사 / 비 - 주입 된 배아를 추적하고 효율적으로 작업 할 수 있도록 상측의 비 주입 배아 드롭의 하측에 삽입 된 것들을 유지한다. </li></ol> 5. 배아 복구 및 사출 결과 <ol><li> 미리 예열 SOF-HEPES로 새로운 접시에 약 200 ㎕를 3 방울을 확인합니다. 마이크로 디스펜서 (위의 설명 참조)를 사용하여 주입 된 배아를 수집합니다. 3 SOF-HEPES를 통해 방울을 이동하여 주입 된 배아를 씻으십시오. </li><li> 미세 주입 중에 용해 배아를 계산하고이를 폐기합니다. 첫 번째 전체 ZP을 작성, 반투명 나타납니다 보낸 용해 접합자는 그대로 하나 쉽게 구별 할 수 있으며, 일반적으로 세포질을 가지고 참고 :배아의 외부로 누출되는 콘텐츠. </li><li> 선택적으로, 형광 현미경을 이용하여 미세 주입 효율을 확인합니다. 자외선 노출이 후속 개발에 영향을 미칠 수있는 배아의 손상을 유도 할 수 있음을 유의하십시오. </li><li> 38.5 ℃에서의 미네랄 오일 아래 4 ㎎ / ㎖ 소 혈청 알부민 (BSA)으로 보충 칼륨 단면 최적화 배지 50㎕의 방울 (KSOM) 25 주입 배아 배양 기, 포화 5 % 공기 중의 CO 2, 습도. </li><li> 이일 주사 후 5 % FBS와 문화 매체를 보충합니다. </li><li> 주사 후 배반포 (BL) 요금 7 일을 확인합니다. 배반포 배아 / 그 드롭 배양 된 배아의 총 수의 총 수와 BL 속도를 계산합니다. </li></ol>

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The authors have nothing to disclose.

접합체의 미세 주입은 포유 동물의 배아에 솔루션을 도입하기위한 잘 정립 된 방법이다. 종 및 실험 목적에 따라 일부 변형이 기술은 광범위하게 사용될 수있다. 우리는 평활 말단 마이크로 피펫의 입구를 돕기 위해 레이저를 사용하여 세포질 마이크로 인젝션을 수행하는 방법을 보여준다. (예 : 소, 양, 돼지 등) 일부 가축 종의 접합자는 배아의 내부에 한 번 주입 피펫의 시각화를 방해, 어두운 ?...

1 셀 배아의 세포질 미세 주입은 매우 강력한 기술이다. 또, 예를 들면, 유전자 기능을 연구 나 유전자 편집 동물을 생성하는 유전자가 녹아웃을 제조 배아에 어떤 솔루션을 제공에 사용될 수있다. 대부분의 농업 관련 농장 동물 접합자는 세포질이 불투명 한 어두운 만드는 매우 높은 지방산 조성. 또한 상당히 탄성 원형질막 (PM)을 갖는다. 이러한 특성은 설치류 종의 도전과 종종 부정확에 사용되는 기존의 전핵 / 세포질 주입을 사용하여 미세 주입을합니다. 이를 수행하기가 쉽습니다 또한 더 높은 생존 능력이 결과, 주입 된 배아에 덜 손상이 발생하기 때문에 세포질 마이크로 인젝션은 전핵 미세 주입을 통해 이점이있다. 이 프로토콜의 전반적인 목표는 농장 동물 접합체의 세포질에 솔루션을 제공하기위한 성공적인 방법을 설명하는 것입니다. 수행 할 수 있어야합니다가축 배아 고효율 세포질 마이크로 인젝션은 레이저는 평활 말단 유리 바늘이 미세 주입에 사용되는 투명대 (ZP) 다음에 구멍을 생성하는데 사용된다. 이 전략은 주입하는 동안 배아에 각인 기계적인 손상을 감소하는 것을 목표로하고있다. 그리고, 주사 바늘 내부 세포질 콘텐츠 흡인 용액 배아의 세포질 내로 전달되는 것을 보장 PM 효율적 자신감 파괴 할 수있다. 이 기술은 이미 성공적 접합체 세포질 3,4-으로 siRNA를 전달하고 클러스터 정기적 interspaced 짧은 상동 반복 (CRISPR) / CRISPR 연관된 시스템 (9) (Cas9) 시스템 (5)을 사용하여 돌연변이를 생성 소 태아에서 사용되어왔다. 또한 소 운 동봉 된 난 모세포 (6)를 주입하기 (약간의 수정과)에 적합하다. 여기서, 우리는 우리의 염료를 제공 주입 프로토콜을 설명하는 데 어떤 주입에 적용 할 수있다수정란 내로 IRED 용액 및이 기술을 사용하여 최소한의 용해가 발생 초기 배아 발달에 영향을주지 않는 것으로 나타났다.

<table border="0" cellpadding="0" cellspacing="0" width="786">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Micropipette puller</td>
			<td style="width:143px;">Sutter Instrument</td>
			<td style="width:119px;">P-97</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Glass capillary</td>
			<td style="width:143px;">Sutter instruments</td>
			<td style="width:119px;">B100-75-10</td>
			<td style="width:257px;">These capillaries are used for making the holding and injecting pipettes. Any thick/standard wall borosilicate tubing without filament can be used.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:119px;">MF-9</td>
			<td>Equipped with 10X magnification lense.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Micromanipulator</td>
			<td style="width:143px;">Nikon/ Narishige</td>
			<td>NT88-V3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Inverted microscope</td>
			<td style="width:143px;">Nikon</td>
			<td>TE2000-U</td>
			<td style="width:257px;">Equipped with 4x, 20x lenses and with a laser system.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser</td>
			<td style="width:143px;">Research Instruments</td>
			<td>7-47-500</td>
			<td>Saturn 5 Active laser.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Microdispenser</td>
			<td>Drummond</td>
			<td>3-000-105</td>
			<td style="width:257px;">The microdispenser is used to move the embryos. A p10 pipette can also be used but loading as minimal volume as possible.</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;width:268px;">60mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:119px;">430166</td>
			<td style="width:257px;">Use the lid of the dish to make the injection plate since they have lower walls and will make positioning and moving of the micropipettes with the micromanipulator easier.&#160;</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;width:268px;">35mm culture dish</td>
			<td style="width:143px;">Corning</td>
			<td style="width:119px;">430165</td>
			<td style="width:257px;">These dishes are used for culturing the embryos in 50&#956;l drops covered with mineral oil. Alternatively, a 4 well dish can also be used. Regardless of the dish chosen to culture the embryos, they always have to be equilibrated in the incubator for at least 4 hours prior to transfering the embryos to them.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Incubator</td>
			<td style="width:143px;">Sanyo</td>
			<td style="width:119px;">MCO-19AIC</td>
			<td style="width:257px;">Any incubator that can be set to 38.5&#176;C 5% CO2 conditions can be used.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">Stereomicroscope</td>
			<td style="width:143px;">Nikon</td>
			<td>SMZ800</td>
			<td style="width:257px;">Used for visualizing the embryos in the culture drops and during washes. Any stereomicroscope with a 10x magnification can be used.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Control Unit HT</td>
			<td style="width:143px;">Minitube</td>
			<td>12055/0400</td>
			<td style="width:257px;">Heating system attached to the stereomicroscope.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heated Microscope Stage</td>
			<td style="width:143px;">Minitube</td>
			<td>12055/0003</td>
			<td style="width:257px;">Heating system attached to the stereomicroscope.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Dextran-Red</td>
			<td>Thermo Scientific</td>
			<td>D1828</td>
			<td>A sterile 10mg/ml solution is used to inject.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:268px;">Mineral Oil</td>
			<td style="width:143px;">sigma</td>
			<td style="width:119px;">M8410</td>
			<td style="width:257px;">Keep the mineral oil at room temperature and&#160; protected from light using foil paper.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">KSOMaa Evolve Bovine</td>
			<td style="width:143px;">Zenit</td>
			<td style="width:119px;">ZEBV-100</td>
			<td style="width:257px;">Supplemented with 4mg/ml BSA. KSOM plates for embryo culture should be equilibrated in an incubator for at least 4 hours before use.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">FBS</td>
			<td style="width:143px;">Gemini-Bio</td>
			<td style="width:119px;">100-525</td>
			<td>Use a stem-cell qualified FBS.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Zygotes</td>
			<td style="width:143px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Zygotes are injected 17-20 hpf and can be in-vitro- or in-vivo-derived.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">NaCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S5886</td>
			<td style="width:257px;">Final concentration: 107.7mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">KCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5405</td>
			<td style="width:257px;">Final concentration: 7.16mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">KH2PO4</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P5655</td>
			<td style="width:257px;">Final concentration: 1.19mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">MgCL2 6H2O</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M2393</td>
			<td style="width:257px;">Final concentration: 0.49mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Sodium DL-lactate</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">L4263</td>
			<td style="width:257px;">Final concentration: 5.3mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">CaCl2-2H2O&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">C7902</td>
			<td style="width:257px;">Final concentration: 1.71mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">D-(&#8722;)-Fructose&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">F3510</td>
			<td style="width:257px;">Final concentration: 0.5mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">HEPES&#160;</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">H4034</td>
			<td style="width:257px;">Final concentration: 21mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">MEM-NEAA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">M7145</td>
			<td style="width:257px;">Final concentration: 1X. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">BME-EAA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">B6766</td>
			<td style="width:257px;">Final concentration: 1X. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">NaHCO3</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">S5761</td>
			<td style="width:257px;">Final concentration: 4mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Sodium pyruvate</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">P4562</td>
			<td style="width:257px;">Final concentration: 0.33mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Glutamax</td>
			<td style="width:143px;">Gibco</td>
			<td style="width:119px;">35050</td>
			<td style="width:257px;">Final concentration: 1mM. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">BSA</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">A-3311</td>
			<td style="width:257px;">Final concentration: 1mg/ml. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:268px;">Gentamicin</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">G-1397</td>
			<td style="width:257px;">Final concentration: 5&#956;g/ml. Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:268px;">Water for embryo transfer</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:119px;">W1503</td>
			<td style="width:257px;">Component of SOF-HEPES medium.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:268px;">SOF-HEPES medium</td>
			<td style="width:143px;">Made in the lab</td>
			<td style="width:119px;"></td>
			<td style="width:257px;">pH 7.3-7.4, 280&#177;10 mOs. Filter sterilized through a 22&#956;m filter can be stored in the fridge at 4&#176; C for 1 month. Warm in 37 &#176;C water bath before use.</td>
		</tr>
	</tbody>
</table>

laser-assisted cytoplasmic microinjection in livestock zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

레이저를 이용한 세포질 미세 주입은 가축 접합체의 세포질에 솔루션을 제공하는 강력하고 신뢰할 수있는 프로토콜입니다. 3 전 및 사출뿐만 아니라 기술의 전반적인 개요 후 접합자의 일반적인 개요를 보여줍니다. 덱스 트란 레드 주입 및 주입 효율 및 정확도를 추적 사이트 있도록 주입 용액으로서 사용된다. 용액의 성공적인 전송은 염료가 균...

Watch this Scientific Journal Video about Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes at JoVE.com

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

레이저를 이용한 가축 접합체의 세포질 미세 주입을

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

laser-assisted-cytoplasmic-microinjection-in-livestock-zygotes

Research

JoVE Journal

Genetics

8.9K 조회수.  University of California, Davis. This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

비디오: Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

갓 고립 된 인간의 산모 Cytotrophoblasts에 siRNA를 형질 및 EMSA 분석

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

연구

유전학

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

세계적인 농업 해충 곤충의 배아 미세 주입을 통해 핵산 배달,<em&gt; Ceratitis의 capitata</em

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

히스톤 - miniSOG에서 사용 Optogenetic 무작위 돌연변이 유발<em&gt; C. 엘레</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

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.

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

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

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 ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene function. Gene knockout has been used to study these gene functions in vivo. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a powerful tool used to edit genomic DNA sequences to alter gene function. While the traditional approach has been to introduce CRISPR/Cas9 mRNA into the single cell embryos through microinjection, this can be a slow and inefficient process in catfish. Here, a detailed protocol for microinjection of channel catfish embryos with CRISPR/Cas9 protein is described. Briefly, eggs and sperm were collected and then artificial fertilization performed. Fertilized eggs were transferred to a Petri dish containing Holtfreter's solution. Injection volume was calibrated and then guide RNAs/Cas9 targeting the toll/interleukin 1 receptor domain-containing adapter molecule (TICAM 1) gene and rhamnose binding lectin (RBL) gene were microinjected into the yolk of one-cell embryos. The gene knockout was successful as indels were confirmed by DNA sequencing. The predicted protein sequence alterations due to these mutations included frameshift and truncated protein due to premature stop codons.

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

A simple and efficient microinjection protocol for gene editing in channel catfish embryos using the CRISPR/Cas9 system is presented. In this protocol, guide RNAs and Cas9 protein were microinjected into the yolk of one-cell embryos. This protocol has been validated by knocking out two channel catfish immune-related genes.

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

태아 Microinjection 및 Nasonia vitripennis 게놈 조작을 위한 이식 기술

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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 ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

Bemisia tabaci Cytogenetic 기술을 사용 하 여 계란 수정 속도 결정

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

급성 골수성 백혈병 세포에서 CRISPR/Cas9 리보뉴클레오단백질에 의한 RUNX1 내성 소음기의 전사 적 역할 조사

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...