Year 2019, Volume 32, Issue 1, Pages 49 - 55 2019-04-01

Chlamydomonas BAC vectörlerinin RED/ET yöntemiyle tek basamakta modifikasyonu
One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system

Münevver AKSOY [1] , Charlene FOREST [2]

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Tüm genom sekansının tamamlanması üzerine, Chlamydomonas BAC’ları fonksiyonel genomik analizlerinde yaygın bir şekilde kullanılmaktadır. Fakat bu vektörlerin hazırlanmalarında kullanılan aşağıdaki sebeplerden dolayı bu tip analizlerde kullanımları optimalden daha düşük olmaktadır: (1) Bu BAC’larda komplementasyon analizlerinde elde edilen transformantların direk seçimini sağlayan bir gen yoktur. (2) BAC ve bağımsız bir vektörün ko-transformasyon (birlikte transformasyon) olarak kullanımında birlikte transformasyon oranı düşüktür. (3) BAC’ların çoğu birden fazla gen içermektedir, bu da her genin farklı bir vektöre sub-klon (alt klonlama) yapılmasını gerektirmektedir (bu da restriksiyon enzimlerine bağımlılık demektir). Bu çalışmada, BAC transformasyonu işlemini kolaylaştırmak için, RED/ET tekniğini kullanarak Chlamydomonas BAC’larına 2 selectable marker cassette (seçilebilir ekspresyon kaseti) eklenmiştir. Bir ökaryotik ve bir prokaryotic ekpresyon kaseti ligasyon edilmiş ve BAC’ların iki basamak modifikasyonu yerine, tek basamakta modifiye edilmesi sağlanmıştır ve bu kasetlerin ekspresyonu kanıtlanmıştır. Böylelikle BAC’ların Chlamydomonas’da fonksiyonel genomik analizlerinde kullanımları için harcanacak zaman süresi azalacaktır.

With the availability of the complete genome, Chlamydomonas BACs are being used extensively in functional genomics analysis. The following aspects of their construction, however, make them less than optimal for some types of analysis. (1) These BACs do not contain a gene to allow direct selection of transformants in complementation analysis. (2) Co-transformation using the BAC and an independent vector with a selectable marker has a low efficiency. (3) Most BACs have more than one gene, necessitating sub-cloning of each gene into a different vector (relying on the use of restriction enzymes). To simplify this process, we modified Chlamydomonas BACs by inserting 2 selectable marker cassettes, using the RED-ET system. We ligated a eukaryotic and a prokaryotic selectable marker cassette and used it in a one-step modification instead of a two-step counter selection protocol and showed the expression of both cassettes. This method will decrease the time needed for use of BACs in functional genomics analysis in Chlamydomonas.


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Primary Language en
Subjects Agricultural, Engineering
Journal Section Makaleler
Authors

Orcid: 0000-0002-0798-5805
Author: Münevver AKSOY (Primary Author)
Institution: Akdeniz University, Faculty of Agriculture
Country: Turkey


Author: Charlene FOREST
Institution: City University of New York, Brooklyn College
Country: United States


Bibtex @research article { mediterranean508032, journal = {Mediterranean Agricultural Sciences}, issn = {}, eissn = {2528-9675}, address = {Akdeniz University}, year = {2019}, volume = {32}, pages = {49 - 55}, doi = {10.29136/mediterranean.508032}, title = {One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system}, key = {cite}, author = {AKSOY, Münevver and FOREST, Charlene} }
APA AKSOY, M , FOREST, C . (2019). One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system. Mediterranean Agricultural Sciences, 32 (1), 49-55. DOI: 10.29136/mediterranean.508032
MLA AKSOY, M , FOREST, C . "One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system". Mediterranean Agricultural Sciences 32 (2019): 49-55 <http://dergipark.org.tr/mediterranean/issue/41736/508032>
Chicago AKSOY, M , FOREST, C . "One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system". Mediterranean Agricultural Sciences 32 (2019): 49-55
RIS TY - JOUR T1 - One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system AU - Münevver AKSOY , Charlene FOREST Y1 - 2019 PY - 2019 N1 - doi: 10.29136/mediterranean.508032 DO - 10.29136/mediterranean.508032 T2 - Mediterranean Agricultural Sciences JF - Journal JO - JOR SP - 49 EP - 55 VL - 32 IS - 1 SN - -2528-9675 M3 - doi: 10.29136/mediterranean.508032 UR - https://doi.org/10.29136/mediterranean.508032 Y2 - 2019 ER -
EndNote %0 Mediterranean Agricultural Sciences One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system %A Münevver AKSOY , Charlene FOREST %T One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system %D 2019 %J Mediterranean Agricultural Sciences %P -2528-9675 %V 32 %N 1 %R doi: 10.29136/mediterranean.508032 %U 10.29136/mediterranean.508032
ISNAD AKSOY, Münevver , FOREST, Charlene . "One step modification of Chlamydomonas reinhardtii BACs using the RED/ET system". Mediterranean Agricultural Sciences 32 / 1 (April 2019): 49-55. https://doi.org/10.29136/mediterranean.508032