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1. Protoplasts 만들기 <ol><li> 페트리 접시에 mannitol 8퍼센트 9 ML을 추가합니다. </li><li> 주걱을 사용하여 5로부터 2-3 PPNH4 접시에서 칠일 오래된 이끼를 넣어 - 페트리 접시가 포함된 mannitol 인치 </li><li> 페트리 접시에 2 % Driselase 3 ML (시그마 D9515 - 25G)을 추가합니다. </li><li> 1 시간 동안 부드러운 잡고 상온에서 페트리 접시를 품어. </li><li> 100 μm의 메쉬 (BD 팰컨 352350)를 통해 원형질의 현탁액을 필터링합니다. </li><li> 5 분 250g에서 필터링된 정지를 스핀. 뜨는을 제거합니다. </li><li> 8 % mannitol 500 μL 매우 부드럽게 protoplasts를 다시 일시 중지합니다. </li><li> 문화 튜브 9.5 ML 8 % mannitol을 추가합니다. protoplasts가 완전히 정지되어 있는지 확인하십시오. </li><li> 여과 다시 정지 단계 (1.6, 1.7 및 1.8) 두 번 더 반복합니다. </li><li> 원형질 솔루션 10 μL를 가지고 hemocytometer에서 protoplasts을 계산합니다. </li><li> (그림 1 참조) ML 당 protoplasts의 숫자를 얻기 위해 10,000의 16 광장 지역에 원형질의 수를 곱하면됩니다. </li></ol> 2. 변환 <ol><li> 5 분 250g의 원형질의 정지를 스핀. 뜨는을 제거합니다. </li><li> 1,600,000 protoplasts / ML의 농도에서 MMG 솔루션 protoplasts를 다시 일시 중지합니다. </li><li> 20 분 상온에서 원형질의 현탁액을 품어. </li><li> 60 μg의 DNA를 포함하는 문화 튜브에 원형질의 정지 600 μL를 추가합니다. 튜브 부드럽게 소용돌이. </li><li> 원형질 / DNA 혼합물에 PEG / CA 솔루션을 700 μL를 추가합니다. 소용돌이는 튜브가 부드럽게 전체 혼합물은 균질 때까지. </li><li> 30 분 상온에서 혼합을 품어. </li><li> 이 대기 기간 동안, 셀로판과 PRM - B 접시 (80mm 직경의 원을, AA 포장 제한, 영국) 커버, 적어도 5 분 플레이트 표면에 수화 셀로판하실 수 있습니다. </li><li> 주걱으로 셀로판와 플레이트 사이에 갇혀있는 공기 방울을 제거하십시오. </li><li> W5 솔루션 3 ML과 혼합물을 희석. </li><li> 5 분 250g의 혼합물을 스핀. 뜨는을 제거합니다. </li><li> PRM - T의 녹은이 ML에서 다시 중지 protoplasts (이 솔루션은 42 ° C에서 액체 유지 수있는 protoplasts에 추가하기 전에). 플레이트 셀로판으로 덮여 PRM - B 플레이트마다 다시 중단 protoplasts 1 ML. Micropore (3M, 의료)와 접시를 넣어. 성장 챔버에서 25 ° C에서 접시 보관하십시오. </li><li> 또는, protoplasts는 액체 도금 매체와 셀로판으로 덮여 PRM - B 접시에 도금 0.5 ML 1 ML에 다시 일시 중지하실 수 있습니다. 이것은 빠른 재생이 가능하지만, 재생 공장의 수가 낮습니다. </li><li> 성장 챔버는 16 시간에 대해 설정됩니다. 빛 8 시간. 어두운주기. </li><li> 사일 변환 후 신선한 선택 접시에에 셀로판를 이동합니다. </li></ol> 3. 대표 결과 : 변환의 예제는 그림 2에 표시됩니다. 이 예제에 사용되는 DNA는 hygromycin 모성 카세트를 들고 플라스미드 supercoiled 7.8 킬로바이트했다. 도금 protoplasts의 양은 사진을 50 %로 축소되었다. 사진은 식물이 선택 접시로 이동했다 2 주 후에 찍은. 변환 효율은 최대 60 μg에 DNA 농도의 영향을받지 않습니다. DNA의 높은 농도는 변화의 효율성에 해로운 있습니다. 데이터는이 방법 DNA 금액의 넓은 범위에서 일관된 결과를 제공하는 것으로 나타났다. 이 방법은 또한 효과적으로 표 1에 나타난 안정적인 transformants를 생성할 수 있으며, 우리는 이전에보고했던 14보다 훨씬 높은 선형 DNA의 마이크로 그램 당 4 안정적인 transformants에 대해받을 수 있습니다. 우리는 또한 마커로서 형광 단백질에 대한 유전자를 운반 supercoiled 플라스미드를 변환하여 변환 효율에 열 충격의 효과를 테스트. 열 충격을 3 분 동안 45 ° C 물 목욕에있는 혼합 잠복기하여 (단계 2.6 이내) 상온 보육 후 10 분 정도 진행되었습니다. 혼합물은 17 분에 대한 열 충격 후 즉시 상온의 물을 욕조에 incubated했다. 결과는 22시간 변환 이후에 기록되었다. 함께 취득 transformants 및 열 충격없이 비율은 (~ 25 %)과 매우 유사했지만 우리는 protoplasts (데이터가 표시되지 않음)의 생존 능력에 열 충격의 부정적인 효과를 관찰했다. <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="그림 1" /> 그림 1. hemocytometer에서 protoplasts의보기. 원형질의 계산에 필요한 부분이 빨간색 사각형으로 표시됩니다. protoplasts를 (1.11 참조) 계산에 필요한 16 사각형을 참고하고, 이미지는 24 protoplasts의 카운트 (240000 셀 / ML)를 보여줍니다. <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="그림 2" /> 그림 2. 십팔일 오래된 과도 transformants 사진. Protoplasts은 트랜스되었습니다supercoiled 플라스미드의 표시 양의 형성. A : 15μg, B : 30μg, C : 60μg, D : 120μg. <table border="1"><tbody><tr><td> DNA (μg)의 금액 </td><td> 효율성 (tranformants / μg의 DNA) </td></tr><tr><td> 15, 플라스미드 supercoiled </td><td> 120 ± 24 </td></tr><tr><td> 30, 플라스미드 supercoiled </td><td> 145 ± 54 </td></tr><tr><td> 60, 플라스미드 supercoiled </td><td> 121 ± 60 </td></tr><tr><td> 120, 플라스미드 supercoiled </td><td> 71 ± 38 </td></tr><tr><td> 60, 플라스미드 선형 </td><td> 4 ± 1 </td></tr></tbody></table> 다양한 DNA 금액에 표 1. 변환 효율.

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	<li>Abel, S., Theologis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Arabidopsis+leaf+protoplasts:+a+versatile+experimental+system+to+study+gene+expression.">Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.</a> Plant J. 5, 421-427 (1994).</li><li>Wood, A. J., Oliver, M. J., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bryophytes+as+model+systems.">Bryophytes as model systems.</a> Bryologist. 103, 128-133 (2000).</li><li>Schaefer, D. G., Zr&#255;d, J. -. 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관심 없음 충돌 선언하지 않습니다.

여기서 제시하는 방법은 Physcomitrella patens의 protoplasts으로 간단하고 일관된 DNA 변환이 가능합니다. 이 방법은 원래 Arabidopsis 1 개발하지만, 여기는 Physcomitrella patens, 간단하고 다른 식물의 protoplasts 잘 수행하는 입증되었다. 따라서,이 방법은 약간의 수정을 다른 식물 수종에 적용할 수 있습니다. 최적화에 대한 가능성이 수정은 MMG의 원형질 농도, MMG와 PEG / 칼슘 용액에 배양 시간을 포함합니다. transformants의 숫자가이 시점 (표 1)까지 D​​NA 농도와 선형 증가하기 때문에 우리는 우리의 일상적인 실험 60 μg의 DNA를 사용하도록 선택할 수 있습니다. 우리는 식물의 대규모 인구의 추가 심사 및 분석을 위해 이용할 수있는 하나의 60 μg의 DNA 변환에 약 7,000 과도 transformants 또는 200 안정적인 transformants를 얻을 수 있습니다. 변형 protoplasts은 액체 도금 매체 또는 PRM - T와 함께 중 전염 수 있습니다. 재생 효율이 액체 도금 매체를 사용하는 경우 낮은이지만 protoplasts가 supercoiled plasmids로 변환했을 때, 우리는이 방법을 선택합니다. 마찬가지로 표 1에 보였다, supercoiled 플라스미드 DNA와 함께 한 변환은 더 많은 transformants를 생성하기 때문에, 우리는 여전히 액체 도금 매체와 식물의 큰 숫자를 얻을 수 있습니다. 또한, 액체 도금 매체와 protoplasts을 전파하는 것은 실험 9,15를 허물고 같은 RNAi과 같은 과도 phenotypes를 관련된 실험을위한 중요합니다 빠른 재생을 허용합니다. 또한 상단 한천의 부재는 immunostaining 및 RT - PCR 9,15 쉽게 공장 절연을 허용합니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 성분 </td></tr><tr><td> PPNH 4 </td><td> 1.84 MM KH 2 PO 4 3.4 MM 칼슘 (NO 3) 2 1 ㎜ MgSO 4 2.72 MM의 Diammonium의 tartrate 54 μm의 FeSO 4 0.7 H 2 O 9.93 μm의 H 3 BO 3 1.97 μm의 MnCl 2 0.4 H 2 O 0.23 μm의 CoCl 2 0.6 H 2 O 0.19 μm의 ZnSO 4. 7H 2 O 0.22 μm의 CuSO 4. 5H 2 O 0.10 μm의 나 2 무 4 0.2 H 2 O 0.168 μm의의 KI 고체 매체에 0.7 %의 한천을 추가합니다. </td></tr><tr><td> PRM - B </td><td> 6 % mannitol과 0.8 %의 한천과 함께 PPNH 4. 접시를 따르고 전에 10 ML 1M CaCl 2-1 패를 추가합니다. </td></tr><tr><td> PRM - T </td><td> 6 % mannitol과 0.6 %의 한천과 함께 PPNH 4. 0.5 ML 1M CaCl 사용하기 전에 2-50 ML을 추가합니다. </td></tr><tr><td> 액체 도금 매체 </td><td> 한천없이 8.5 %의 mannitol과 PPNH 4. 0.5 ML 1M CaCl 사용하기 전에 2-50 ML을 추가합니다. </td></tr><tr><td> 2 % Driselase </td><td> Driselase 4 G (시그마 D9515 - 25G) 8 % mannitol 200 ML에 추가합니다. 30 분 부드럽게 저어. 상온에서 30 분 알을 품다. 4 ° C, 5 분 부드럽게 저어. 상온에서. 펠렛을 삭제, 10 분 2천5백그램를 스핀. 10 ML aliquots 얼어붙은 0.22 μm의, 그리고 저장과 필터. 사용하기 전에 실온의 물을 욕조에 녹여. </td></tr><tr><td> MMG </td><td> 0.4 M mannitol 15 MM MgCl 2 4 MM MES (산도 5.7) </td></tr><tr><td> PEG / CA </td><td> 4g PEG4000 3 ML H 2 O 2.5 ML 0.8 M mannitol 1 ML 1M CaCl 2 </td></tr><tr><td> W5 </td><td> 154 MM NaCl 125 MM CaCl 2 5 MM KCl 이 MM MES (산도 5.7) </td></tr></tbody></table>

efficient polyethylene glycol (peg) mediated transformation of the moss physcomitrella patens

<p class="jove_content"변환하기&gt; 간단하고 효율적인 방법<em&gt; Physcomitrella pantens</em&gt; protoplasts이 설명되어 있습니다. 이 방법은을 위해 프로토콜에서 적응됩니다<em&gt; Physocmitrella</em&gt; protonemal 원형질과<em&gt; Arabidopsis</em&gt; mesophyll의 원형질 변환<sup&gt; 1</sup&gt;. 상동 재조합 효율 mitotic를 받아야하기 위해 용량으로 인해<em&gt; Physcomitrella patens</em&gt;은 최근에 중요한 모델 시스템으로 등장했습니다<sup&gt; 2</sup&gt;. 이 용량은 유전자의 높은 주파수 타겟팅 가능<sup&gt; 3-9</sup같은 다른 모델 식물을 볼 수 없습니다&gt;<em&gt; Arabidopsis</em&gt;. 이 시스템을 최대한 활용하기 위해, 우리는 이끼 세포에 DNA를 제공하기 위해 효과적이고 쉬운 방법이 필요합니다. 이 이끼를 변환하는 가장 일반적인 방법은 입자 충격 아르<sup&gt; 10</sup&gt; 그리고 PEG - 중재 DNA 이해<sup&gt; 11</sup&gt;. 입자 폭격 높은 변환 효율을 생산할 수 있지만<sup&gt; 12</sup&gt;, 유전자 총을 많은 실험실에게 즉시 제공되지 않으며 프로토콜은 표준화하기가 어렵습니다. 한편, PEG 중재 변화 전문 장비를 필요로하지 않으며, 살균 후드가있는 모든 실험실에서 수행할 수 있습니다. 여기, 우리는 모스 protoplasts의 변환을위한 간단하고 매우 효율적인 방법을 보여줍니다. 이 방법은 이전에 보고된 가장 효율적인 프로토콜의 개선입니다 DNA의 마이크로 그램 당 120여 과도 transformants를 생성할 수 있습니다<sup&gt; 13</sup&gt;. 간단, 효율성 및 재현성 때문에,이 방법은 일상적인 변화뿐만 아니라 transformants의 다수를 요구하는 프로젝트에 적용할 수 있습니다.</p

Watch this Scientific Journal Video about Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens at JoVE.com

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

변환하는 간단하고 효율적인 방법<em&gt; Physcomitrella pantens</em&gt; protoplasts이 설명되어 있습니다. 이 방법은을 위해 프로토콜에서 적응됩니다<em&gt; Physocmitrella</em&gt; protonemal 원형질과<em&gt; Arabidopsis</em&gt; mesophyll의 원형질 변환<sup&gt; 1</sup&gt;.

모스의 효율적인 폴리에틸렌 글리콜 (PEG) 중재 변환 Physcomitrella patens</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

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Biology

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

40.5K 조회수.  Worcester Polytechnic Institute- WPI. 변환하는 간단하고 효율적인 방법 Physcomitrella pantens protoplasts이 설명되어 있습니다. 이 방법은을 위해 프로토콜에서 적응됩니다 Physocmitrella protonemal 원형질과 Arabidopsis mesophyll의 원형질 변환 1.

비디오: Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

플렉스의 변경을 물려 전할 수있는 환경을 유도

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also ...

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생물학

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

에틸렌 글리콜 기반 Vitrification에 의한 마우스 태아의 Cryopreservation

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

Picoeukaryote의 게놈 변환<em&gt; Ostreococcus 타우 리</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

재조합 단백질의 높은 수준의 과도 발현 식물의 효율적인 Agroinfiltration

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

에피 솜과 HDAC 억제제를 사용하여 혈액에서 효율적인 유도 만능 줄기 세포 생성

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

아그로박테리움-형태유전자를 이용한 리칼시트 옥수수 근친선 미성숙 배아 변형 중재

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

CRISPR/Cas9를 사용하여 Null 돌연변이를 생성하기 위해 컬렉스 모기의 배아를 준비하고 주입합니다.

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

Agrobacterium-매개 유전 변환, 트랜스 제닉 생산, 그리고 쌀에서 남성 생식 개발의 연구에 대 한 그것의 응용 프로그램

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

가을 육군벌레, 스포드옵테라 검소한에서 활동적인 피기박 트랜스포사스 중재 된 세균라인 변환

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

자연 형질전환, 단백질 발현 및 필라멘트성 시아노박테리움 포미듐 라쿠나의 동결보존(Cryoconservation of Filamentous Cyanobacterium Phormidium lacuna)

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...

모스의 효율적인 폴리에틸렌 글리콜 (PEG) 중재 변환 Physcomitrella patens

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