이 출판물은 Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica (PAPIIT), UNAM [IN223623 to XP-M]의 지원을 받았습니다. UPD는 CONAHCYT 펠로우(CVU:883299)입니다. 광학 현미경 이미지에 대한 기술적 도움을 주신 Ariann Mendoza-Martínez 박사님께 감사드립니다. Biorender 라이선스: DU26OMVLUU(그림 2); BK26TH9GXH(그림 3); GD26TH80R5(그림 4); PU26THARYD(그림 7); ML26THAIFG(그림 9).

참고: 미토콘드리아 변환 효율은 일반적으로 낮기 때문에 각 구조에 대해 6개의 변환을 수행하는 것이 좋습니다. 상이한 성장 매체의 조성은 표 2에 나타내어져 있다.
1. 텅스텐 입자 제조
<ol><li>마이크로튜브에서 0.7μm 텅스텐 입자(WP, 마이크로캐리어) 30mg을 칭량합니다. 1.5% 에탄올(EtOH) 70mL를 첨가하여 살균합니다. WP를 소용돌이치게 하고 실온에서 10분 동안 그대로 둡니다.</li>
	<li>실온에서 13,200 x g 에서 15분 동안 원심분리합니다. 입자에 1.5mL의 멸균수를 첨가하고 소용돌이치게 한 다음 실온에서 13,200× g 에서 15분 동안 원심분리하여 WP를 세척합니다. 상층액을 버립니다.</li>
	<li>500μL의 멸균 50% 글리세롤을 추가합니다(최종 농도는 WPs/μL 60μg). 알림: WP는 -20 °C에서 최소 6개월 동안 보관할 수 있습니다. 이 스톡에서 6 회 변형에 필요한 양 (100 μL)을 취하십시오.</li>
</ol>2. WPs DNA 코팅
<ol><li>미토콘드리아 서열을 포함하는 2 μ plasmid 5 μg과 박테리아 plasmid 15 μg을 얼음 위의 마이크로튜브에 추가합니다. 참고: DNA 코팅에 필요한 시약을 더 희석하지 않도록 최종 부피는 50μL를 초과해서는 안 됩니다.</li>
	<li>100μL의 WP 현탁액과 5초 동안 와류를 추가합니다(WP 6mg).</li>
	<li>1M 스퍼미딘 4μL를 추가합니다. 간단히 말해서 소용돌이.</li>
	<li>100 μL의 얼음처럼 차가운 2.5 M CaCl2 를 넣고 잠시 와류를 넣으십시오. 얼음 위에서 10분 동안 배양합니다. 3분마다 믹스를 짧게 소용돌이칩니다. 알림: CaCl2 용액은 필터 멸균 처리되어 있으며 고압멸균해서는 안 됩니다.</li>
	<li>4°C에서 13,200× g 의 WP를 30초 동안 원심분리하고 상층액을 버립니다. -20°C에서 200μL의 100% EtOH에 입자를 부드럽게 재현탁하여 세척합니다.</li>
	<li>4°C에서 13,200× g 의 WP를 30초 동안 원심분리하고 상층액을 버립니다. 세탁을 반복하십시오.</li>
	<li>피펫 팁을 사용하여 60-70 μL의 실온 100% EtOH에 WP를 재현탁시킵니다. 소용돌이치지 마십시오. 참고: 플라스미드의 품질을 보장하기 위해, DNA 코팅은 biolistic transformation과 같은 날에 수행되어야 합니다.</li>
</ol>3. 생물학적 재료 준비
<ol><li>폭격하기 전에 오토클레이브에서 6개의 마크로캐리어 홀더(그림 5)를 멸균하고 완전히 건조시킵니다. 또는 EtOH에 담그고 겸자를 사용하여 화염을 사용하여 살균합니다. 멸균 유리판에 놓습니다.</li>
	<li>DNA 침전 중(2.4단계에서 얼음 위에서 10분 배양) 멸균 겸자를 사용하여 6개의 마크로캐리어 홀더 각각에 6개의 마크로캐리어(또는 플라잉 디스크)를 삽입합니다. 알림: 매크로캐리어를 멸균하지 마십시오.</li>
	<li>DNA 코팅된 WP를 피펫과 혼합합니다. 각 매크로캐리어 표면에 10μL의 WP를 추가하고 피펫 팁으로 전체 표면에 분포시킵니다. 필요한 경우 100% EtOH의 몇 마이크로리터를 매크로캐리어 표면에 추가하여 WP가 고르게 퍼지도록 합니다. 현탁액을 실온에서 ~20분 동안 건조시키십시오.</li>
</ol>4. 효모 세포 및 충격판의 준비
<ol><li>YPR 배지 2mL에 rho0 수용체 균주를 접종합니다(표 2). 회전하는 바퀴에서 30 °C에서 하룻밤 동안 성장시킵니다.</li>
	<li>배양액 300μL를 신선한 YPR 배지 30mL에 희석합니다. 30 °C 및 200 rpm에서 로터리 셰이커로 2박 동안 배양액이 포화될 때까지 성장시킵니다.</li>
	<li>효모 세포를 600× g 에서 실온에서 5분 동안 원심분리합니다. 상층액을 버리고 600μL의 YPD 배지에 효모 세포를 재현탁시킵니다(표 2).</li>
	<li>멸균 유리 손잡이를 사용하여 100μL의 효모 세포 현탁액을 고체 폭격 배지 플레이트에 펴 바릅니다. NOTE: bombardment medium에는 transformation에 사용될 2 μ plasmid의 selection marker가 없어야 합니다.</li>
	<li>다른 5개의 플레이트에 대해 4.4단계를 반복합니다.</li>
	<li>액체 매체가 플레이트 표면에 완전히 흡수되도록 충격하기 전에 실온에서 1-3시간 동안 배양합니다.</li>
</ol>5. 생물학적 형질전환에 의한 합성 rho-균주의 생성
알림: 시작하기 전에 생물학적 장비의 사용 설명서를 반드시 읽으십시오. 참조된 장비의 프로토콜에 대해 설명합니다(재료 표 및 그림 5).
<ol><li>오염을 방지하기 위해 70% 에탄올로 생물학적 입자 전달 시스템의 챔버를 청소하고 최소 10분 동안 그대로 두십시오. 폭격 직전에 깨끗한 종이 타월로 표면을 닦으십시오.</li>
	<li>헬륨 탱크 밸브를 열어 약 2,000psi를 설정합니다.</li>
	<li>장비의 전원을 켜고 진공 소스를 켭니다.</li>
	<li>집게를 사용하여 매크로캐리어를 매크로캐리어 홀더에 아래를 향하게 놓습니다. 알림: 장비와 함께 제공되는 정지 화면을 사용하지 마십시오.</li>
	<li>매크로캐리어 위에 매크로캐리어 뚜껑을 나사로 고정합니다. 모든 부품이 함께 마이크로캐리어 발사 시스템을 구성합니다(그림 5).</li>
	<li>마이크로캐리어 발사 시스템을 바닥에서 다섯 번째 레벨의 진공 챔버 내부에 놓습니다.</li>
	<li>rho0 변형 잔디를 포함하는 플레이트 중 하나를 챔버 바닥에서 두 번째 레벨에 놓습니다. 접시가 위를 향하고 뚜껑이 없는지 확인하십시오.</li>
	<li>멸균 겸자를 사용하여 고정 캡에 하나의 파열 디스크를 놓습니다. 가속 튜브의 고정 캡을 조입니다. 고정 캡이 꼭 맞는지 확인하십시오. 알림: 변형 효율이 감소하는 것을 관찰한 결과 설명서에서 제안한 대로 파열된 디스크를 이소프로판올로 처리하지 마십시오.</li>
	<li>진공 챔버의 문을 닫습니다.</li>
	<li>VAC/VENT/HOLD 스위치를 VAC 위치에 놓습니다. 25-28 inHg 진공에 도달하면 VAC/VENT/HOLD 스위치를 HOLD 위치로 변경합니다. 알림: 달성 가능한 진공은 해수면에 따라 달라질 수 있습니다. 우리는 25 inHg에 도달하는 데 성공했습니다.</li>
	<li>FIRE 스위치를 눌러 WP를 쏘십시오. 파열 디스크가 파손되어 팝 소리가 날 때 1,300psi에 도달할 때까지 가속 튜브에 압력이 형성될 때까지 기다립니다.</li>
	<li>파열 디스크가 파손된 직후 VAC/VENT/HOLD 스위치를 VENT 위치로 변경하여 FIRE 스위치와 진공을 해제합니다.</li>
	<li>멸균 겸자를 사용하여 플레이트 표면에 있는 파열 디스크의 파편을 조심스럽게 제거하고 플레이트를 뚜껑으로 덮습니다.</li>
	<li>나머지 5개의 플레이트에 대해 절차를 반복하여 각 라운드에 새 파열 디스크와 매크로캐리어를 배치합니다.</li>
	<li>마지막에 헬륨 탱크의 메인 밸브를 닫습니다.</li>
	<li>챔버를 닫고 VAC/VENT/HOLD 스위치를 VAC 위치로 가져와 진공을 만듭니다. FIRE 스위치를 몇 번 눌렀다 놓아 헬륨 압력에서 시스템을 해제합니다. 문을 연 상태에서 반복합니다.</li>
	<li>진공 펌프와 폭격 장비를 끕니다.</li>
	<li>70% EtOH로 전달 시스템을 청소합니다. 매크로캐리어 지지대를 0.1% SDS 용액으로 세척하십시오. 멸균 증류수와 100% EtOH로 헹굽니다.</li>
</ol>6. 합성 rho- 콜로니 선택
<ol><li>30 °C에서 5-7 박 동안 폭격 플레이트를 배양합니다. 알림: 플레이트에 오염이 있는지 매일 확인하는 것이 중요합니다. 필요한 경우 추가 오염을 방지하기 위해 멸균 주걱으로 오염된 부위를 잘라낼 수 있습니다.</li>
	<li>반대쪽 결합 유형의 테스터 균주를 밤새 YPD 배지 2mL에 접종합니다(그림 2D,E의 특정 예 참조).</li>
	<li>YPD 플레이트에 150μL의 테스터 균주 배양액을 접종합니다. 멸균 유리 손잡이를 사용하여 테스터를 플레이트 표면에 고르게 펴 바릅니다. 접시를 최소 5분 동안 건조시키십시오.</li>
	<li>벨벳 복제 패드와 복제기 스탬프를 사용하여 폭격판의 복제품을 만드십시오.<ol><li>형질전환된 2 μ plasmid auxotrophy marker에 해당하는 dropout medium plate에 복제합니다. 성장은 30 °C에서 한 번의 하룻밤 배양이 필요합니다. 그 후 4 °C에서 계속 사용할 때까지 보관하십시오. 이 플레이트가 마스터 플레이트입니다.</li>
		<li>6.3단계에서 테스터의 잔디밭이 포함된 YPD 플레이트에 복제합니다. 30°C에서 2박 동안 배양합니다. 그 후, 이 플레이트를 선택적 배지에 복제하여 관심 미토콘드리아 유전자(예: 호흡기 배지 또는 아르기닌이 결핍된 배지)의 존재를 검출하고 30°C에서 밤새 배양합니다. 미토콘드리아에 합성 박테리아 플라스미드를 품고 있는 세포만이 테스터 균주와 교배한 후 선택적 배지에서 자랍니다.</li>
	</ol></li>
	<li>마스터 플레이트(단계 6.4.1)에서 단계 6.4.2에서 성장한 해당 콜로니를 식별합니다. 알림: 폭격 플레이트(및 마스터 플레이트)는 변형 콜로니로 붐비기 때문에 테스터의 결합 플레이트에서 관찰된 양성 콜로니를 식별하기 어려울 수 있습니다. 이러한 이유로 영구 마커로 모든 판의 양쪽에 몇 개의 작은 선을 그리는 것이 좋습니다. 복제 스탬프의 링에 비슷한 선을 그립니다. 이 선은 마스터 플레이트에서 양극 콜로니의 방향을 지정하고 위치를 찾는 데 도움이 됩니다( 그림 2D의 예 참조).</li>
	<li>6.4.1단계에서와 같이 마스터 플레이트에서 두 번째 선택적 드롭아웃 플레이트로 줄무늬를 만들어 양극 콜로니를 정제합니다. 30 °C에서 2박 동안 배양합니다. 참고 : 줄무늬는 순수한 변형을 보장하기 위해 단일 식민지의 성장을 허용하는 것이 중요합니다.</li>
	<li>선택 과정(단계 6.2-6.5)을 반복하여 미토콘드리아에 박테리아 플라스미드가 있는지 확인합니다. 참고: 일반적으로 이 과정을 두 번 더 반복하여 안정적인 합성 rho-strain 을 선택할 수 있습니다.</li>
	<li>합성 rho-train 이 미토콘드리아에서 플라스미드 DNA를 안정적으로 유지하면 3-5개의 서로 다른 양성 콜로니를 선택하고 2mL의 YPD에 접종합니다. 이 배양액을 사용하여 cryovial stock을 준비하고(600μL의 배양액과 600μL의 50% 글리세롤을 혼합하여 -70°C에서 보존) 세포유도를 통해 플라스미드 염기서열을 관심 mtDNA에 삽입합니다(단계 6.7). 주의: 합성 rho-strain 으로부터 미토콘드리아에서 형질전환된 플라스미드를 유지하기 위한 선택적 압력이 없기 때문에, 플라스미드는 쉽게 손실될 수 있습니다. 따라서 가능한 한 빨리 세포 유도를 하는 것이 좋습니다.</li>
</ol>7. 세포유도에 의해 미토콘드리아 게놈의 표적 부위에 합성 rho-train에 존재하는 구조를 통합합니다.
<ol><li>두 개의 튜브에 3mL의 YPD 배지를 접종하는데, 하나는 공여 세포(합성 rho- 세포)로, 다른 하나는 수용체 세포(최종 구조가 예상되는 효모 세포)로 접종합니다. 30 °C에서 하룻밤 동안 배양합니다.</li>
	<li>750 μL의 donor 균주와 250 μL의 수용체 균주를 멸균 마이크로튜브(3:1 비율)에 혼합합니다.</li>
	<li>혼합물을 13,200 × g에서 1 분 동안 원심 분리하십시오. 상층액을 버리십시오. 끝이 잘린 1mL 피펫 팁을 사용하여 세포 펠릿 한 방울을 YPD 플레이트에 놓습니다. 30 °C에서 2-4 시간 동안 배양합니다. 알림: 교차 오염을 방지하려면 단일 플레이트에 두 개 이상의 셀 현탁액을 넣지 마십시오(예:그림 4A의 파일).</li>
	<li>소량의 세포 혼합물을 취하여 슬라이드의 15μL의 멸균수에 재현탁시키고 커버 슬립을 놓습니다. 광학 현미경으로 40x 또는 100x 대물렌즈로 shmoos가 있는지 확인하십시오. 이 특징적인 모양은 두 가지 결합 유형이 있을 때 나타납니다(그림 3B).</li>
	<li>shmoos가 존재하는 경우 2mL의 YPD 배지에 소량(~0.5mm3)의 세포 질량을 재현탁시킵니다. 회전하는 휠에서 30°C에서 2시간 동안 배양하여 이핵 접합체가 회복될 수 있도록 합니다.</li>
	<li>혼합물을 와류시키고 세포 현탁액 10μL를 멸균수 990μL에 희석합니다(1:100 희석). 유리 구슬 또는 멸균 유리 손잡이를 사용하여 수용체 균주(및 산발적인 이배체)만 성장하는 드롭아웃 배지에 세포 희석액 100μL를 펴 바릅니다. 30 °C에서 2-3 박 동안 배양합니다.</li>
	<li>서론에서 설명한 대로 생성된 콜로니를 필요한 선택적 매체에 복제합니다(4단계, 특정 예는 그림 4C 참조).</li>
	<li>양성 콜로니를 식별한 후 동일한 선택적 매체에 다시 스트리크하여 관심 mtDNA를 운반하는 반수체 균주를 정제합니다.</li>
	<li>두 개 이상의 양성 콜로니를 선택하고 30°C의 YPD 배지 2mL에서 밤새 성장시킵니다. 600μL의 50% 글리세롤과 600μL의 포화 YPD 배양물을 혼합하여 극저온 백업을 만듭니다. -70 °C에서 보관하십시오.</li>
	<li>생성 된 균주31,32의 DNA를 분리합니다. 미토콘드리아 돌연변이의 존재를 확인하기 위해 PCR과 염기서열로 원하는 영역을 증폭합니다. 참고: 각 양성 콜로니는 미토콘드리아 게놈에서 올바른 염기서열을 보장하기 위해 독립적으로 확인해야 하는 기술적 복제입니다.</li>
</ol>

돌연변이 유전 분석을 위한 효모 미토콘드리아의 생물학적 형질전환

<ol>
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저자는 공개할 이해 상충이 없습니다.

본 연구는 효모 S. cerevisiae 에서 미토콘드리아를 성공적으로 변형시키는 방법을 설명했습니다. 고속 미세 발사체 충격에서 의도 된 효모 균주의 정제까지의 과정은 합성 rho-train 의 정제 횟수에 따라 8 ~ 12 주가 소요됩니다. 이 방법의 중요한 단계 중 일부는 다음과 같습니다. 첫째, 미토콘드리아 유전자 구조에서 돌연변이 부위 주변에 추가되는 측면 영역이 클수록 표적 돌연변이의 ?...

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL pipette tips</td>
			<td>Axygen</td>
			<td>T-1000-B</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Microtube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#956;L pipette tips</td>
			<td>Axygen</td>
			<td>T-10-C</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical bottom tube&#160;</td>
			<td>Axygen</td>
			<td>SCT-25ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L pipette tips</td>
			<td>Axygen</td>
			<td>T-200-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical bottom&#160; tube</td>
			<td>Axygen</td>
			<td>SCT-50ML-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>AfiII</td>
			<td>New England BioLabs</td>
			<td>R0520S</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>SeaKem</td>
			<td>50004</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytic balance</td>
			<td>OHAUS</td>
			<td>ARA520</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td>TOMY</td>
			<td>ES-315</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
		</tr>
		<tr>
			<td>Biolisitic Macrocarrier holder&#160;</td>
			<td>BIO-RAD</td>
			<td>1652322</td>
			<td></td>
		</tr>
		<tr>
			<td>Bunsen burner</td>
			<td>VWR</td>
			<td>89038-528</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -ADE</td>
			<td>Formedium</td>
			<td>DCS0049</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -ARG</td>
			<td>Formedium</td>
			<td>DCS0059</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -LEU</td>
			<td>Formedium</td>
			<td>DCS0099</td>
			<td></td>
		</tr>
		<tr>
			<td>CSM -URA</td>
			<td>Formedium</td>
			<td>DCS0169</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture glass flask</td>
			<td>KIMAX KIMBLE</td>
			<td>25615</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture glass tube</td>
			<td>Pyrex</td>
			<td>9820</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>BD</td>
			<td>215520</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>JT Baker&#160;</td>
			<td>9000</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Millipore</td>
			<td>620006</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>Z265926</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass handle</td>
			<td>Sigma</td>
			<td>S4647</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>JT BAKER</td>
			<td>2136-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Helium tank grade 5 (99.99 %)</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>HSTaq&#160; Kit</td>
			<td>PCR BIO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifugue</td>
			<td>Eppendorf</td>
			<td>022620100</td>
			<td></td>
		</tr>
		<tr>
			<td>NdeI</td>
			<td>New England BioLabs</td>
			<td>R0111L</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>New Brunswick scientific</td>
			<td>NB-G25</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Axygen</td>
			<td>PCR-02-C</td>
			<td></td>
		</tr>
		<tr>
			<td>PDS-1000/He TM Biolistic Particle Delivery System</td>
			<td>BIO-RAD</td>
			<td>165-2257</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes (100X10)</td>
			<td>BD</td>
			<td>252777</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>Raffinose</td>
			<td>Formedium</td>
			<td>RAF03</td>
			<td></td>
		</tr>
		<tr>
			<td>Replica plater</td>
			<td>Scienceware</td>
			<td>Z363391</td>
			<td></td>
		</tr>
		<tr>
			<td>Rupture discs 1350 Psi</td>
			<td>BIO-RAD</td>
			<td>1652330</td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbitol</td>
			<td>Sigma</td>
			<td>S7547</td>
			<td></td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA Ligase</td>
			<td>Thermo Scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Rotator</td>
			<td>Thermo Scientific</td>
			<td>88882015</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten microcarriers M10</td>
			<td>BIO-RAD</td>
			<td>1652266</td>
			<td></td>
		</tr>
		<tr>
			<td>Vaccum pump of 100L/min capacity</td>
			<td>-</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Velvet pads</td>
			<td>Bel-Art</td>
			<td>H37848-0002</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex&#160;</td>
			<td>Scientifc Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wood aplicator stick</td>
			<td>PROMA</td>
			<td>1820060</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen base without aminoacids</td>
			<td>BD</td>
			<td>291920</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial transformation in baker&#39;s yeast to study translation and respiratory complex assembly

베이커 효모 맥주효 모균(Saccharomyces cerevisiae )은 수십 년 동안 미토콘드리아 생물학을 이해하는 데 널리 사용되어 왔습니다. 이 모델은 진핵생물 사이에서 필수적이고 보존된 미토콘드리아 경로와 곰팡이 또는 효모 특이적 경로에 대한 지식을 제공했습니다. S. cerevisiae 의 많은 능력 중 하나는 미토콘드리아 게놈을 조작하는 능력으로, 지금까지 S. cerevisiae 와 단세포 조류 Chlamydomonas reinhardtii에서만 가능합니다. 효모 미토콘드리아의 생물학적 형질전환(biolistic transformation)을 통해 부위 지정 돌연변이(site-directed mutation)를 소개하고, 유전자를 재배열하고, 리포터(reporter)를 소개할 수 있습니다. 이러한 접근법은 주로 미토콘드리아에서 고도로 조정된 두 가지 과정, 즉 미토리보솜에 의한 번역과 호흡기 복합체 및 ATP 합성효소의 조립의 메커니즘을 이해하는 데 사용됩니다. 그러나 미토콘드리아 변환은 잠재적으로 다른 경로를 연구하는 데 사용될 수 있습니다. 본 연구에서는 고속 미세발사체 충격에 의해 효모 미토콘드리아를 형질전환시키는 방법, 의도한 형질전환체를 선별 및 정제하는 방법, 원하는 돌연변이를 미토콘드리아 게놈에 도입하는 방법을 보여줍니다.

The yeast Saccharomyces cerevisiae is a widely recognized model used to study mitochondrial biogenesis. Since yeast is an anaerobic, facultative organism, it is possible to extensively study the causes and consequences of introducing mutations that impair respiration. In addition, this organism possesses friendly genetic and biochemical tools to study mitochondrial pathways. However, one of the most powerful resources to explore the mechanisms of respiratory complex assembly and mitochondrial protein synthesis is the ability to transform mitochondria and modify the organelle&#39;s genome. Previously, it has been helpful to introduce in the mitochondrial DNA (mtDNA) point mutations or small deletions/insertions1,2,3,4,5, delete genes6,7, make gene rearrangements7,8, add epitopes to mitochondrial proteins9,10, relocate genes from the nucleus to the mitochondria11,12, and introduce reporter genes like BarStar13, GFP14,15, luciferase16, and the most widely used ARG8m17,18. Mitochondrial genome modifications have allowed us to dissect and identify mechanisms that otherwise would have been difficult to comprehend. For example, the ARG8m reporter gene inserted at the COX1 locus&#160;in the mitochondrial DNA was crucial to understanding that Mss51 has a dual role in Cox1 biogenesis. First, it is a translational activator of the COX1 mRNA, and second, it is an assembly chaperone for the newly made Cox1 protein7,19. This work presents a detailed method to transform S. cerevisiae mitochondria. Although the mitochondrial transformation protocol was published earlier16,20,21,22,23, a visual approach through a video is essential to thoroughly understand the different stages and details of the method. The method consists of various steps and is divided into four general stages:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66856/66856fig01v2.jpg" /> 
Figure 1: Overview of the mitochondrial transformation procedure by high-velocity microprojectile bombardment: 1) The tungsten particles are sterilized and prepared before DNA coating. 2) Two different plasmids are precipitated on the surface of the WPs. One is a bacterial plasmid containing the construct that will be directed to the mitochondrial matrix. The other is a nuclear yeast expression vector carrying an auxotrophy marker. 3) A receptor yeast strain lacking mitochondrial DNA (rho0) is grown on a fermentative carbon source like galactose or raffinose, which does not exert any glucose repression of mitochondrial gene expression34,35. The culture is spread on petri dishes containing the bombardment medium. 4) WPs coated with plasmids are shot to the receptor strain by high-velocity microprojectile bombardment. 5) Positive synthetic rho- colonies containing the mitochondrial plasmid are selected by mating to a tester strain. 6) The mitochondrial construct is integrated into the mitochondrial genome at the desired locus by mating the synthetic rho- strain (donor) with the acceptor strain by a process known as cytoduction. 7) The positive receptor, haploid strain carrying the mitochondrial construct is selected and purified in different media. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Transformation of cells with the DNA construct intended to integrate into the mitochondrial genome (Figure 1, steps 1-4) 
Although it is possible to directly transform a yeast containing a complete mitochondrial genome (rho+), the transformation is 10-20x more efficient if the cells lack mitochondrial DNA (rho0)24. Tungsten particles (WPs) are coated with two different plasmids that will be co-transformed. The first one is a yeast 2 &#181; expression vector carrying an auxotrophy marker, such as LEU2 or URA3 (e.g., YEp351 or YEp352, respectively). If the biolistic introduction of DNA into the yeast cells is successful, the transformants will grow on the auxotrophy medium (i.e., a medium lacking leucine or uracil). This plasmid is helpful in making the first selection of the cells that acquired the nuclear plasmid; otherwise, the number of resulting colonies would saturate the plate. The second plasmid is a bacterial plasmid (such as pBluescript or similar) containing the mitochondrial construction intended to be integrated into the mitochondrial genome. The construct must contain at least 100 nt of 5&#39; and 3&#39; flanking mitochondrial sequences for recombination with the mitochondrial region of interest. In our experience, larger flanking sequences are more likely to successfully recombine with the target locus in the mitochondrial genome.
A specific example is shown in Figure 225. In this example, the aim was to delete the region of the mitochondrial COX1 gene coding for the last 15 amino acids of the corresponding protein (Cox1&#916;C15) (Figure 2A). The bacterial plasmid carrying the COX1&#916;C15 mutation contained 395 nt and 990 nt of the gene&#39;s 5&#39; and 3&#39; untranslated regions, respectively. The plasmid was derived from pBluescript (pXPM61) and, together with the 2 &#181; plasmid YEp352, they were coprecipitated on the surface of WPs (Figure 2B). The WPs were then introduced by microprojectile bombardment into the selected recipient strain (Figure 2C). This strain, named NAB6926, is a MATa, rho0 strain bearing the kar1-1 and ade2 alleles&#160;(the importance of these characteristics is discussed below). The cells were plated on bombardment media lacking uracil to select those that acquired the 2 &#181; plasmid (Figure 2C). Cells were grown for 5-7 nights at 30 &#176;C.
Selection of the cells that acquired the bacterial plasmid containing the mitochondrial construct and the nuclear 2 &#181; plasmid carrying the auxotrophy marker (Figure 1, step 5) 
The positive colonies will maintain many copies of the bacterial plasmid in their mitochondria22. Since the transformed cells are originally rho0, no mitochondrial sequences support the translation of the mitochondrial construct present in the plasmid; hence, the transformed mitochondrial gene will not be expressed. It is necessary to mate the transformants with a tester strain to detect the yeast colonies that acquired the mitochondrial plasmid. The mitochondrial genome of the tester strain contains a nonfunctional mutated version of the gene of interest. After mating, the mitochondria will fuse, and the mitochondrial sequence included in the transformed plasmid will recombine with the mutated mitochondrial gene from the tester strain; consequently, the recovery of the WT gene will reconstitute function. The resulting diploid will have a detectable positive phenotype (usually the capacity to grow in respiratory medium or a medium lacking arginine). The positive cells carrying the bacterial plasmid in mitochondria are named &#34;synthetic rho- cells&#34;. In the specific example of Figure 2D,&#160;synthetic rho- cells carrying the COX1&#916;C15 construct (named XPM199) were replica-plated on a medium lacking uracil (this is the master plate from which positive colonies were purified). They were also replica-plated on a tester strain L4527 lawn, which contains the nonfunctional mutation cox1D369N. After mating for two nights, the mitochondrial genome of L45 and XPM199 recombined, resulting in a functional COX1 gene; therefore, the diploids recovered the ability to grow on respiratory media. From the master plate, we picked the positive colonies. They were streaked on plates lacking uracil, and the selective tests were repeated to obtain pure synthetic rho- cells (Figure 2E). It is important to note that the testing plate is only for the identification of synthetic rho- colonies, and mutants cannot be recovered from these plates.
Integration of the construct into the mitochondrial genome of the intended strain 
This step is achieved by a process called cytoduction28,29 (Figure 1, step 6). In this approach, the synthetic rho- strain (donor strain) is mated to the intended acceptor strain of the opposite mating type. An essential requirement is that at least one of the two mating strains carries the kar1-1 mutation to impede nuclear fusion30. Hence, mating of the two cells produces a binucleated zygote (Figure 3). The mitochondrial network from the parental cells (donor and acceptor) fuse and the mitochondrial DNA molecules recombine. The binucleated zygotes are incubated/recovered to allow budding, which will produce haploid cells. These haploids carry the nuclear background of one of the parental cells. Similarly, haploids can carry the mitochondrial DNA from either the parental cell or the recombined mitochondrial DNA of interest. However, the kar1-1 mutation is not 100% effective, and some true diploids will be formed during the mating28,29. The synthetic rho- strain (donor, XPM199) carried the kar1-1 allele in the example. As a selective marker, it had the ade2 allele, making it an auxotroph for adenine (Figure 4). The acceptor strain was XPM10, a rho+ strain bearing the cox1&#916;::ARG8m&#160;construct, where the reporter ARG8m&#160;replaced the COX1 codons in the mitochondrial genome7. The mixture of both cell cultures was added to a YPD plate as a drop to allow mating. After 3-5 h, the cells were observed under a light microscope to detect the formation of shmoos (Figure 3B), a cell shape associated with mating. After the incubation/recovery time, the binuclear zygotes were plated on a medium lacking adenine to prevent the growth of the donor cells.
Selection of the haploid strain carrying the mitochondrial genome of interest (Figure 1, step 7) 
A mixture of donor, acceptor, and diploid cells is present after cytoduction. Therefore, after the incubation/recovery of the binucleated zygotes, cells must be grown in different selective media to identify and purify the haploids of interest. The selective media depends on the genotypes of the donor, acceptor strains, and the intended mitochondrial construction. However, in general, the reasoning for choosing the selective media is: i) after incubation/recovery, the cytoduction mix is grown on selective medium where the donor parental strain cannot grow. In the specific example in Figure 4A,B, the donor (synthetic rho- strain) carries the nonfunctional ade2 allele. Thus, the cytoduction mix must be incubated on a medium plate lacking adenine. This is the master plate. ii) Once the colonies grow, the master plate is replicated again on a medium lacking adenine (to generate a new master plate from where the positive colonies of interest will be purified), and next, on a medium where only diploids can grow. This is necessary to avoid further inadvertent purification of diploid colonies. In the case of the example from Figure 4C, only diploids can grow on media lacking leucine. iii) The master plate is also replicated on media where only those acceptor haploids that incorporated the mitochondrial DNA of interest will grow. In the example of Figure 4C,&#160;haploids grow on respiratory media containing ethanol/glycerol as a carbon source since the Cox1&#916;C15 protein is functional. The resulting rho+ strain bearing the mtDNA of interest was named XPM20925. The strains used in Figure&#160;2 and Figure 4 are listed in Table 1.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66856/66856fig02.jpg" /> 
Figure 2: Diagram describing a specific example of mitochondrial transformation and selection of positive rho- cells. (A) The intended modification of the mitochondrial genome was a deletion of the region coding for the last 15 amino acids of the Cox1 subunit (Cox1&#916;C15). (B) WPs were coated with two plasmids to transform yeast cells. One was the 2 &#181; plasmid Yep352 that was directed and expressed in the nucleus. The other was plasmid pXPM61, which contains the COX1&#916;C15 allele plus 395 nt and 990 nt of the COX1 5&#39; and 3&#39;-UTRs, respectively. (C) The WPs were introduced into cells from the strain NAB69, which lacks mitochondrial DNA (rho0 strain). Transformant colonies obtained from the bombardment plates were replicated on a medium lacking uracil, the auxotrophic marker of the nuclear plasmid YEp352. (D) To select the positive rho- colonies, the -URA plate was replicated on a media lacking uracil. This was the master plate from which the positive colonies were picked and isolated. It was also replicated on plates with rich media (YPD) and a lawn of a tester strain. During mating, the synthetic rho- mitochondrial DNA was recombined with the mitochondrial DNA from the tester strain, L4527, which carries a mutation in the COX1 gene (D369N). The diploids that grew on respiratory media contained the transformed DNA. The corresponding colonies were picked from the master plate to restreak and purify. To help identify the positive colonies in the master plate throughout all replica plating, marks were made with a permanent marker on the edges of the plates (green lines). (E) The selected positive colonies were restreaked on a medium lacking uracil. This was the new master plate. As in D, two more rounds of testing were carried out to purify the synthetic rho- colonies. Abbreviations: WPs = tungsten particles; mtDNA = mitochondrial DNA; -URA/Sorb = lacking uracil/ containing Sorbitol;&#160;WT = wild type. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66856/66856fig03v2.jpg" /> 
Figure 3: Diagram describing the cytoduction procedure. (A) General overview of how cells mate during cytoduction.&#160;During cytoduction, mitochondria from the donor and acceptor strains fuse so mitochondrial DNA from both strains recombine. Since nuclear fusion is reduced due to the kar1-1 mutation30,&#160;binuclear zygotes are formed. After some incubation/recovery time, the binuclear zygotes bud and haploid acceptors containing the intended mitochondrial mutation are selected. (B) Mating of the synthetic rho- strain (donor) and the acceptor strain through cytoduction allows the formation of shmoos (red arrows), a characteristic shape of mating yeast. The image was taken under a light microscope with a 100x objective. Scale bar = 10 &#181;m. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig03largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66856/66856fig04.jpg" /> 
Figure 4: Diagram describing a specific example of cytoduction to integrate the mutation COX1&#916;C15 in the mitochondrial genome. (A) Liquid cultures of the synthetic rho- strain (donor, XPM199) and the strain of interest (acceptor, XPM10) were mixed to mate. After shmoo formation, the mix was recovered in a liquid culture for 2-4 h. Next, the cytoduction mix was spread on a plate with a medium lacking adenine to prevent the growth of the donor cells. (B) Schematic representation of the recombination events of the mitochondrial DNA from the donor (XPM199) and the acceptor (XPM10) during cytoduction. (C) The master plate was replicated on different selective media to identify and purify those haploids that integrated the intended mitochondrial gene in the organelle&#39;s DNA (XPM209)25. Abbreviations: -ADE = lacking adenine; -LEU = lacking leucine. <a href="https://www.jove.com/files/ftp_upload/66856/66856fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

저자 스포트라이트: 혁신적인 미토콘드리아 연구를 통한 단백질 합성 및 조립의 기술 및 발견 발전

베이커의 미토콘드리아 변환

Our research explores the regulation of mitochondrial protein synthesis and its connection to inner membrane assembly, crucial for oxidative phosphorylation. Using the model, we study the molecular mechanism of mitochondrial function, leveraging its suitability for detailed molecular and genetic investigations. Many technologies are used for research in our field, like RNA deep sequencing, multiomic technologies, cryo-electron microscopy, high-velocity bombardment to transform gene mitochondria, among others.We have made important contributions to understand the role and mechanisms of action of many proteins involved in mitochondrial translation regulation and on respiratory complexes III and IV assembly. This is the case for proteins like Mss51, Pet309, Pet54, mS38, CPP3, CPP6, among others. This protocol is an indispensable tool for understanding how mitoribosomes find the STARD-CHOL mitochondrial mRNAs.It has been the key to understanding the role of specific nucleotides of mitochondrial mRNAs in translation regulation. These are some examples of what this technique can address. The only available resource to mitochondrial sequences and to use translation of the in the mitochondrial DNA is our protocol, high-velocity bombardment transformation of gene mitochondria.This method has provided invaluable information regarding translation and respiratory complexes assembly that otherwise we could not obtain.

이 섹션에서는 미토콘드리아 변형의 여러 단계에서 얻은 몇 가지 대표적인 결과를 제시합니다. 그림 6은 폭격 절차를 보여줍니다. 합성 rho-cell은 미토콘드리아 유전자의 암호화 서열을 대체할 리포터 유전자 ARG8m을 가진 박테리아 플라스미드를 운반했습니다(그림 6A). 폭격 후, 플레이트는 우라실(-URA)이 결핍된 매체에 복제되었습...

이 연구는 생물학적 방법을 사용하여 효모 미토콘드리아를 형질전환하는 방법을 설명합니다. 또한 형질전환체를 선택하고 정제하는 방법과 미토콘드리아 게놈 내의 표적 위치에 원하는 돌연변이를 도입하는 방법을 보여줍니다.

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about ...

mitochondrial-transformation-baker-s-yeast-to-study-translation

Research

JoVE Journal

Genetics

Mitochondrial Transformation in Baker&#39;s Yeast to Study Translation and Respiratory Complex Assembly

477 조회수.  Universidad Nacional Autónoma de México. 우리의 연구는 미토콘드리아 단백질 합성의 조절과 산화적 인산화에 중요한 내막 조립과의 연결을 탐구합니다. 이 모델을 사용하여 미토콘드리아 기능의 분자 메커니즘을 연구하고 상세한 분자 및 유전 연구에 대한 적합성을 활용합니다. RNA 심층 염기서열 분석, 다중체 기술, 초저온 전자 현미경, 유전자 미토콘드리아를 변형시키기 위한 고속 폭격?...

비디오: 저자 스포트라이트: 혁신적인 미토콘드리아 연구를 통한 단백질 합성 및 조립의 기술 및 발견 발전

Baker&#180;s yeast Saccharomyces cerevisiae has been widely used to understand mitochondrial biology for decades. This model has provided knowledge about essential, conserved mitochondrial pathways among eukaryotes, and fungi or yeast-specific pathways. One of the many abilities of S. cerevisiae is the capacity to manipulate the mitochondrial genome, which so far is only possible in S. cerevisiae and the unicellular algae Chlamydomonas reinhardtii. The biolistic transformation of yeast mitochondria allows us to introduce site-directed mutations, make gene rearrangements, and introduce reporters. These approaches are mainly used to understand the mechanisms of two highly coordinated processes in mitochondria: translation by mitoribosomes and assembly of respiratory complexes and ATP synthase. However, mitochondrial transformation can potentially be used to study other pathways. In the present work, we show how to transform yeast mitochondria by high-velocity microprojectile bombardment, select and purify the intended transformant, and introduce the desired mutation in the mitochondrial genome.

This work explains how to transform yeast mitochondria using a biolistic method. We also show how to select and purify the transformants and how to introduce the desired mutation in the target position within the mitochondrial genome.

연구

유전학

Material Preparation for Biolistic Transformation of Yeast 

To begin, add 1.5 milliliters of 70%ethanol to 30 milligrams of 0.7-micrometer tungsten particles in a microtube. Centrifuge the mixture at 13, 200G for 15 minutes. Add 1.5 milliliters of sterile water to the particles, then vortex and centrifuge again.After discarding the supernatant, add 500 microliters of sterile 50%glycerol to the tungsten particles. To coat the particles, first add five micrograms of a two-micron plasmid and 15 micrograms of a bacterial plasmid containing the mitochondrial sequence into a microtube placed on ice. Add 100 microliters of the tungsten particle suspension to the tube and vortex.Then add four microliters of one-molar spermidine and vortex again. Now pipette 100 microliters of ice cold 2.5-molar calcium chloride into the tube. After briefly vortexing the suspension, incubate it on ice for 10 minutes.Centrifuge the tungsten particles at 13, 200G for 30 seconds at four degrees Celsius. Then, wash the particles in 200 microliters of 100%ethanol at minus 20 degrees Celsius. Finally, re-suspend the tungsten particles in 60 to 70 microliters of room temperature 100%ethanol.To prepare the biolistic materials, place six sterilized macrocarrier holders on a sterile glass plate. Use a pair of sterile forceps to insert the macrocarriers into each of the six macrocarrier holders. Pipette 10 microliters of the DNA-coded tungsten particles onto each macrocarrier surface and evenly distribute over the entire surface with the pipette tip.Inoculate the rho zero yeast receptor strain in two milliliters of YPR medium. Grow the culture overnight at 30 degrees Celsius in a rotating wheel. The next day, transfer 300 microliters of the culture into a tube containing 30 milliliters of fresh YPR medium.Place the culture on a rotary shaker for two nights at 30 degrees Celsius and 200 revolutions per minute until the culture is saturated. Centrifuge the yeast cells at 600G for five minutes at room temperature. Re-suspend the cells in 600 microliters of YPD medium after discarding the supernatant.Now use a sterile glass handle to spread 100 microliters of the yeast cell suspension on a solid bombardment medium plate. After spreading all the cultures, incubate the plates at room temperature for one to three hours before bombardment.

효모의 생물학적 변형을 위한 재료 준비 

Generation of a Synthetic Yeast Rho- Strain by Biolistic Transformation for Mutagenetic Studies

To begin, add 70%ethanol to the chamber of the biolistic particle delivery system. After 15 minutes, wipe the ethanol with a clean paper towel. Open the helium tank valves to set a pressure of 2, 000 pounds per square inch.Next, use a pair of sterile forceps to place a rupture disc on the retaining cap, and screw the retaining cap on the acceleration tube. Use a pair of forceps to place a macrocarrier facing down on the macrocarrier holder, then screw the macrocarrier lid on top of the macrocarrier. Place the microcarrier launch system inside the vacuum chamber at the fifth level from the bottom.Then, place a plate containing the rho zero strain lawn on the second level from the bottom of the chamber. Put the vacuum vent hold switch in the vacuum position. When the vacuum reaches 20 to 21 inches of mercury, change the vacuum vent hold switch to the hold position.Press the fire switch to shoot the tungsten particles. When the pressure reaches 1, 300 pounds per square inch, the rupture disc will break, making a pop sound. Immediately after the rupture disc breaks, change the vacuum vent hold switch to the vent position to release the fire switch and the vacuum.Use sterile forceps to carefully remove any debris from the rupture disc present on the surface of the plate. Then cover the plate with its lid.

돌연변이 유전학 연구를 위한 생물학적 변형에 의한 합성 효모 Rho- 균주의 생성

Selection of Yeast Synthetic Rho- Colonies for Mutagenetic Studies

To begin, incubate the biolistic bombardment plates of yeast at 30 degrees Celsius for five to seven nights. Inoculate the tester strain of the opposite mating type in two milliliters of YPD medium overnight. The next day, transfer 150 microliters of the tester strain culture on a YPD plate.With a sterile glass handle, spread the tester evenly on the plate's surface and let it air dry. Use velvet replicating pads and a replicator stamp to make a master plate replica of the transformed plasmid auxotrophy marker on drop-out medium plate. Then, replicate on the YPD plate containing the tester's lawn and incubate.After 48 hours, replicate the plate containing the tester on a selective medium to detect the presence of the mitochondrial gene of interest. Compare the master plate and the corresponding colonies to identify the positive colonies. Streak the positive colonies from the master plate onto a second selective drop-out plate.The tester lawn and the transformant cells mated and resulted in infused mitochondria. Purified positive synthetic rho-colonies were obtained from URA plates. Some positive synthetic rho-colonies lost the mitochondrial construct.

돌연변이 유전학 연구를 위한 효모 합성 Rho- 콜로니의 선택

Integration of the Construct Present in the Synthetic Rho- Strain into the Yeast Mitochondrial Genome by Cytoduction

To begin, inoculate two tubes containing three milliliters of YPD media with the synthetic rho minus cells and the acceptor yeast cells. The next day, transfer 750 microliters of the donor strain and 250 microliters of the receptor strain into a sterile microtube. After centrifuging the mixture for one minute at 13, 200G, transfer a drop of the cell pellet onto a YPD plate and incubate.Check the culture under a light microscope for the presence of shmoos. If shmoos are present, re-suspend a small amount of the cell mass in two milliliters of YPD medium. Incubate the culture for two hours at 30 degrees Celsius in a rotating wheel.Vortex the mixture well. Then dilute 10 microliters of the cell suspension in 990 microliters of sterile water. Spread 100 microliters of the diluted culture on a drop-out medium that facilitates the growth of only the acceptor strain.Replicate the resulting colonies on the necessary selective media. After identifying positive colonies, re-streak them on the same selective media to purify the haploid strain carrying the mitochondrial DNA of interest.