LV NSF (IOS 1.002.837) tarafından desteklenen

1. Protoplast yapma <ol><li> Petri kabı mannitol% 8 9 ml ekleyin. </li><li> 5 2-3 PPNH4 plakalardan bir spatula kullanarak, 7 gün eski yosun koymak Petri kabı içeren mannitol. </li><li> Petri kabı 3 ml (Sigma D9515-25G) 2% Driselase ekleyin. </li><li> Petri kabı, 1 saat süreyle hafif sallayarak ile oda sıcaklığında inkübe edin. </li><li> 100 mikron mesh (BD Falcon 352.350) ile protoplast süspansiyon Filtresi. </li><li> 5 dakika boyunca 250 gr süzülmüş süspansiyon dönerler. Süpernatantı. </li><li> 500 mcL% 8 mannitol ile çok yavaşça protoplast yeniden askıya. </li><li> Kültür tüpüne 9.5 ml% 8 mannitol ekleyin. Protoplast tamamen askıya alınır emin olun. </li><li> Filtrasyon ve yeniden süspansiyon adımları (1.6, 1.7 ve 1.8) iki kez daha tekrarlayın. </li><li> Protoplast çözüm 10 mcL alın ve bir hemasitometre protoplast saymak. </li><li> (Bkz: Şekil 1) mL başına protoplast numarası almak için 16 kare alanda protoplast sayısı 10.000 ile çarpın. </li></ol> 2. Dönüşüm <ol><li> 5 dakika için 250 g protoplast süspansiyon Spin. Süpernatantı. </li><li> 1,6 milyon protoplast / mL konsantrasyonda MMG çözüm protoplast yeniden askıya. </li><li> Protoplast süspansiyon 20 dakika oda sıcaklığında inkübe edin. </li><li> Protoplast süspansiyon 60 mg DNA içeren bir kültür tüpe 600 mcL ekleyin. Tüp nazikçe Swirl. </li><li> 700 PEG / Ca çözüm mcL protoplast / DNA karışımı ekleyin. Swirl tüp nazikçe tüm karışımı homojen kadar. </li><li> Karışımı oda sıcaklığında 30 dakika inkübe edin. </li><li> Bu bekleme süresi sırasında, PRM-B selofan levhalar (80 mm çapında daireler, AA Ambalaj Limited Şirketi, Birleşik Krallık) kapak; levha yüzeyi üzerinde en az 5 dakika hidrat izin selofan. </li><li> Spatula ile, selofan ve plaka arasında sıkışmış hava kabarcıkları. </li><li> W5 çözüm 3 mL karışım sulandırınız. </li><li> Spin karışımı 5 dakika boyunca 250 gr. Süpernatantı. </li><li> PRM-T eritilmiş 2 mL yeniden askıya protoplast (bu çözüm, 42 ° C&#39;de sıvı muhafaza edilebilir protoplast eklemeden önce). Plaka PRM-B selofan kapsadığı plaka başına yeniden askıya protoplast 1 ml. Micropore (3M Sağlık) plakaları sarın. Bir büyüme odasında plakalar 25 ° C&#39;de tutun. </li><li> Alternatif olarak, 1 ml sıvı kaplama orta ve selofan ile kaplanmış bir PRM-B plaka kaplama 0,5 mL protoplast yeniden askıya alınmış olabilir. Bu hızlı yenilenmesini sağlar, ancak yenileyici bitkilerin sayısı düşüktür. </li><li> Büyüme odası, 16 saat için ayarlanır. ışık ve 8 saat. karanlık döngüsü. </li><li> Selofan dönüşüm 4 gün sonra yeni bir seçim plaka üzerine taşıyın. </li></ol> 3. Temsilcisi Sonuçlar: Dönüşüm bir örnek Şekil 2&#39;de gösterilmiştir. Bu örnekte kullanılan DNA hygromycin dayanıklı kaset taşıyan plazmid supercoiled 7.8 kb oldu. Fotoğrafçılığı için% 50, kaplama protoplast miktarı azaldı. Resimler bitkilerin seçimi plakaları taşındı iki hafta sonra alındı. Dönüşüm verimliliği DNA konsantrasyonu 60 mg kadar etkilenmez. DNA yüksek konsantrasyonlarda dönüşüm verimliliği zararlıdır. Bu yöntem veri DNA miktarda geniş bir yelpazede içinde tutarlı sonuç sağladığını gösterdi. Bu yöntem aynı zamanda etkili bir şekilde Tablo 1&#39;de gösterilmiştir istikrarlı transformants oluşturabilir, daha önce 14 bildirilmiştir çok daha yüksek Lineerleştirilmiş DNA mikrogram başına 4 istikrarlı transformants alabilirsiniz. Biz de bir belirteç olarak flüoresan protein geni taşıyan bir supercoiled plazmid dönüştürerek dönüşüm verimliliği ısı şok etkisi test edilmiştir. Isı şoku, 3 dakika boyunca 45 ° C su banyosu karışımı kuluçka (Adım 2.6 içinde), oda sıcaklığında kuluçka 10 dakika sonra gerçekleştirildi. Karışım 17 dakika süreyle ısı şok hemen sonra oda sıcaklığında su banyosunda inkübe edildi. Sonuçlar 22 saat sonra dönüşüm kaydedildi. Transformants ile elde edilen ve ısı şok olmadan oranları (~% 25) çok benzer, ama biz protoplast (veriler gösterilmemiştir) canlılığı üzerindeki ısı şok bir yan etki gözlenmedi. <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Şekil 1" /> Şekil 1. Hemasitometre içinde protoplast gör. Protoplast sayımı için gerekli bölüm, kırmızı bir kare ile gösterilir . Not protoplast (1.11) sayımı için gerekli olan 16 kareler; görüntü 24 protoplast sayısı (240.000 hücre / ml). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Şekil 2" /> Şekil 2. 18 günlük geçici transformants Resimleri protoplast transsupercoiled plazmid belirtilen miktarda kurdu. A: 15μg; B: 30μg; C: 60μg; D: 120μg. <table border="1"><tbody><tr><td> DNA (mikrogram) Miktarı </td><td> Verim (tranformants / mg DNA) </td></tr><tr><td> 15, plazmid supercoiled </td><td> 120 ± 24 </td></tr><tr><td> 30, plazmid supercoiled </td><td> 145 ± 54 </td></tr><tr><td> 60, plazmid supercoiled </td><td> 121 ± 60 </td></tr><tr><td> 120, plazmid supercoiled </td><td> 71 ± 38 </td></tr><tr><td> 60, plazmid lineerleştirilmiş </td><td> 4 ± 1 </td></tr></tbody></table> Tablo 1: Çeşitli DNA miktarlarda Dönüşüm verimliliği.

<ol>
	<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. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+in+the+moss+Physcomitrella+patens.">Efficient gene targeting in the moss Physcomitrella patens.</a> Plant J. 11, 1195-1206 (1997).</li><li>Schaefer, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+in+Physcomitrella+patens.">Gene targeting in Physcomitrella patens.</a> Curr. Opin. Plant Biol. 4, 143-150 (2001).</li><li>Kamisugi, Y., Cuming, A. C., 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=Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens.">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Res. 33, e173-e173 (2005).</li><li>Cove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moss+Physcomitrella+patens.">The moss Physcomitrella patens.</a> Annu. Rev. Genet. 39, 339-358 (2005).</li><li>Kamisugi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+gene+targeting+in+Physcomitrella+patens:+homologous+recombination,+concatenation+and+multiple+integration.">The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.</a> Nucleic Acids Res. 34, 6205-6215 (2006).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+formin-mediated+actin-filament+elongation+is+essential+for+polarized+plant+cell+growth.">Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.</a> Proc. Natl. Acad. Sci. U.S.A. , (2009).</li><li>Sawahel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+DNA+into+Physcomitrella+protonemal+tissue+by+using+the+gene+gun.">Transfer of foreign DNA into Physcomitrella protonemal tissue by using the gene gun.</a> Plant Mol. Biol. 10, 315-316 (1992).</li><li>Schaefer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+the+moss+Physcomitrella+patens.">Stable transformation of the moss Physcomitrella patens.</a> Mol. Gen. Genet. 226, 418-424 (1991).</li><li>Cho, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+bombardment+mediated+transformation+and+GFP+expression+in+the+moss+Physcomitrella+patens.">Particle bombardment mediated transformation and GFP expression in the moss Physcomitrella patens.</a> Mol. Cells. 9, 14-19 (1999).</li><li>Ashton, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bryophyte+Physcomitrella+patens+replicates+extrachromosomal+transgenic+elements.">The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements.</a> New Phytol. 146, 391-402 (2000).</li><li>Hohe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+highly+standardized+transformation+procedure+allows+efficient+production+of+single+and+multiple+targeted+gene-knockouts+in+a+moss,+Physcomitrella+patens.">An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.</a> Curr. Genet. 44, 339-347 (2004).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+is+essential+for+tip+growth+in+the+moss+Physcomitrella+patens.">Profilin is essential for tip growth in the moss Physcomitrella patens.</a> Plant Cell. 19, 3705-3722 (2007).</li></ol>

Burada sunulan yöntem Physcomitrella Dini ayinlerde kullanılan patenler protoplast içine basit ve tutarlı bir DNA dönüşümü sağlar . Bu yöntem başlangıçta Arabidopsis 1 için geliştirilmiş, ancak burada Physcomitrella Dini ayinlerde kullanılan patenler, daha basit ve farklı bitki protoplast ile iyi bir performans ortaya koydu. Bu nedenle, bu yöntem, küçük değişiklikler ile diğer bitki türlerinin uygulanabilir. Optimizasyonu için olası değişiklikler MMG protoplast konsantrasyon, kuluçka süresi, MMG ve PEG / Ca çözüm içerir. (Tablo 1) Bu noktada transformants sayısı kadar DNA konsantrasyonu ile doğrusal olarak artar, çünkü bizim rutin deneyler 60 mg DNA kullanmayı tercih. Biz bitkilerin büyük bir nüfus daha fazla tarama ve analiz için izin veren tek bir 60 mg DNA dönüşümü, yaklaşık 7000 geçici transformants veya 200 istikrarlı transformants alabilir. Dönüştürülmüş protoplast sıvı kaplama, orta ya da PRM-T ile iki yayılmış olabilir. Protoplast supercoiled plazmid dönüştürülmüş olduğu rejenerasyon sıvı kaplama orta kullanıldığında verimliliği düşük olmasına rağmen, bu yöntemi tercih etmektedir. Tablo 1&#39;de gösterdi supercoiled plazmid DNA ile yapılan dönüşümler, çok daha fazla transformants ve bu nedenle, biz hala sıvı kaplama orta ile çok sayıda bitki elde edebilirsiniz. Ayrıca, sıvı kaplama orta protoplast yayılmasını deneyler 9,15 yıkmak, RNAi olarak geçici fenotipleri içeren deneyler için önemli olan hızlı yenilenme sağlar. Buna ek olarak üst agar yokluğunda, immün ve RT-PCR 9,15 kolay bitki izolasyon sağlar .

<table border="1"><tbody><tr><td> Reaktifin Adı </td><td> Bileşen </td></tr><tr><td> PPNH 4 </td><td> 1.84 mM KH 2 PO 4 3.4 mM Ca (NO 3) 2 1 mM MgSO 4 2.72 mM Diamonyum tartarat 54 mcM FeSO 4 .7 H 2 O 9.93 mcM H 3 BO 3 1.97 mcM MnCl 2 .4 H 2 O 0.23 mcM CoCl 2 .6 H 2 O 0.19 mcM ZnSO 4. 7H 2 O 0.22 mcM CuSO 4. 5H 2 O McM 0.10 Na 2 Moo 4 .2 H 2 O 0.168 mcM KI Katı besiyeri için% 0.7 agar ekleyin. </td></tr><tr><td> PRM-B </td><td> PPNH 4,% 6 mannitol ve% 0.8 agar . Plakalar dökülmeden önce 10 mL 1M CaCl 2 1 L ekleyin. </td></tr><tr><td> PRM-T </td><td> PPNH 4,% 6 mannitol ve% 0.6 agar . 0.5 mL 1M CaCl kullanmadan önce 2 ila 50 ml ekleyin. </td></tr><tr><td> Sıvı Kaplama Orta </td><td> PPNH 4% 8.5 agar olmadan mannitol. 0.5 mL 1M CaCl kullanmadan önce 2 ila 50 ml ekleyin. </td></tr><tr><td> 2% Driselase </td><td> Driselase 4 g (Sigma D9515-25G),% 8 mannitol 200 ml ekleyin. 30 dakika hafifçe karıştırın. oda sıcaklığında 30 dakika inkübe edin. 4 ° C ve 5 dakika boyunca yavaşça karıştırın. oda sıcaklığında. Pelet atın, 10 dakika Spin 2.500 gr. Filtre 10 ml alikot olarak dondurulmuş 0.22 mikron ve mağaza. Kullanmadan önce oda sıcaklığında su banyosunda çözülme. </td></tr><tr><td> MMG </td><td> 0,4 M mannitol 15 mM MgCl 2 4 mM MES (pH 5.7) </td></tr><tr><td> PEG / Ca </td><td> 4 g 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 MES (pH 5.7) 2 mM </td></tr></tbody></table>

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

Physcomitrella pantens protoplast dönüştürmek için basit ve etkili bir yöntem açıklanmıştır. Bu yöntem Physocmitrella protonemal protoplast ve Arabidopsis mezofil protoplast dönüşüm 1 protokolleri uyarlanmıştır. Verimli mitotik homolog rekombinasyon geçmesi kapasitesi nedeniyle Physcomitrella Dini ayinlerde kullanılan patenler, son yıllarda 2 önemli bir model sistem olarak ortaya çıkmıştır . Bu kapasite, Arabidopsis gibi diğer model tesislerinde görülen 3-9 hedef gen yüksek frekansları sağlar . Bu sistemin tam olarak yararlanmak için, biz yosun hücrelerin içine DNA sunmak için etkin ve kolay bir yöntem gerekir. Bu yosun dönüştürmek için en yaygın yolu, partikül bombardımanı 10 ve PEG-aracılı DNA alımı 11 . Partikül bombardımanı yüksek dönüşüm verimliliği 12 üretebilir rağmen, gen silahlar birçok laboratuvarda hazır değildir ve protokol standardize etmek zordur. Öte yandan, PEG aracılı dönüşüm uzman ekipman gerektirmeyen, ve steril bir kukuleta ile, herhangi bir laboratuvar yapılabilir. Burada, yosun protoplast dönüşüm için basit ve son derece etkili bir yöntem gösteriyor. Bu yöntem daha önce 13 DNA, en verimli protokolü bir gelişmedir mikrogram başına 120&#39;den fazla geçici transformants üretebilir . Basitlik, verimlilik ve tekrarlanabilirlik için, bu yöntem rutin dönüşümün yanı sıra çok sayıda transformants gerektiren projeler için uygulanabilir.

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

Dönüştürmek için basit ve etkili bir yöntem<em&gt; Physcomitrella pantens</em&gt; Protoplast açıklanmıştır. Bu yöntem için protokolleri uyarlanmıştır<em&gt; Physocmitrella</em&gt; Protonemal protoplast ve<em&gt; Arabidopsis</em&gt; Mezofil protoplast dönüşüm<sup&gt; 1</sup&gt;.

Moss Verimli Polietilen Glikol (PEG) aracılığı ile Dönüşüm Physcomitrella Dini ayinlerde kullanılan patenler</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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Research

JoVE Journal

Biology

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

40.3K Görüntüleme.  Worcester Polytechnic Institute- WPI. Dönüştürmek için basit ve etkili bir yöntem Physcomitrella pantens Protoplast açıklanmıştır. Bu yöntem için protokolleri uyarlanmıştır Physocmitrella Protonemal protoplast ve Arabidopsis Mezofil protoplast dönüşüm 1.

Video: 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.

Keten içinde değişiklikler Kalıtsal Çevre İndüklenmiş

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

Biyoloji

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.

Etilen Glikol Tabanlı Vitrifikasyon Fare Embriyolar Kriyoprezervasyon

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.

Picoeukaryote genomik Dönüşüm<em&gt; Ostreococcus tauri</em

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

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.

Agrobacterium-Rekalsitrant Mısır Inbred Hatları Morfojenik Genler Kullanarak Aracılı Olgunlaşmamış Embriyo Dönüşümü

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/Cas9 Kullanarak Boş Mutasyonlar Oluşturmak için Culex Sivrisineklerinin Embriyolarının Hazırlanması ve EnjekteSi

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-Aracılı Genetik Dönüşüm, Transgenik Üretim ve Pirinçte Erkek Üreme Gelişimi Çalışmaları Için Uygulaması

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.

Hiperaktif piggyBac Transposase aracılı Germline Dönüşümü Sonbahar Ordu Kurdu, Spodoptera frugiperda

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

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the &#946;-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.

Agrobacterium tumefaciens-Dar Yapraklı Muzun Aracılı Genetik Transformasyonu

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack ...

Kökle İlgili Biyolojiyi İlerletmek için Tek Adımlı Tüylü Kök Dönüşüm Yöntemi

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Yazar Gündemi: Agrobacterium rhizogenes Aracılı Tüylü Kök Dönüşümü ile Kompozit Bitki Üretiminin Kolaylaştırılması 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Agrobacterium rhizogenes Bazlı Tüylü Kök Dönüşümü ile Kompozit Bitkilerin Üretilmesi İçin Verimli ve Tekrarlanabilir Bir Yöntem

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...