Dear all,
I just thought about marker free transgenic crop plants when i was cloning a gene (the gene which i work on) in plant expression vector that we recently bought in our lab. The thought was to generate plant with vector merely having desired gene, promoter, terminator and of course, the DNA region required for integration. It should not have any marker genes like hygromycin, kanamycin and even reporter genes such as GUS, GFP. The question came in my mind is that why do we need to use all these genes to screen transgenic plants when we have primers.
Are those marker genes going to enhance transformation frequency?
Is that laborious or not possible to generate transgenic plants with out having selection markers?
I welcome your comments and suggestions.
Friday 16 December 2011
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8 comments:
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I guess we should confine
Marker-free transgenics are quite possible. There have been a lot of technical papers on this since 2005 mostly revolving around Cre/Lox recombination and its improvements.
Hi jegan,
As you said, only problem comes in selection. It is worth to screen more plants using PCR rather selecting plants with marker genes (‘unwanted genes’ with regard to generation of transgenic plants).My suggestion is that let grow the plants till regeneration and screen for transgenic plant using PCR. I would say, it is not laborious because all we wanted to have marker free transgenic plants and it will serve the purpose and there is also no question of safety aspect of transgenic plants.
I would also say, screening the plants using PCR is cost effective method. All antibiotics what we use for selection are very costly (Approximately 5 grams of hygromycin-85,000 Rs).
Hi jegan,
As you said, only problem comes in selection. It is worth to screen more plants using PCR rather selecting plants with marker genes (‘unwanted genes’ with regard to generation of transgenic plants).My suggestion is that let grow the plants till regeneration and screen for transgenic plant using PCR. I would say, it is not laborious because all we wanted to have marker free transgenic plants and it will serve the purpose and there is also no question of safety aspect of transgenic plants.
I would also say, screening the plants using PCR is cost effective method. All antibiotics what we use for selection are very costly (Approximately 5 grams of hygromycin-85,000 Rs).
hi all,
I can agree with marker free transgenics but screening the transgenic plants by PCR is not easy.
Because of the low efficiency of transgene integration , we will get more non-transformed plant than the transformed one. screening of transgenic line among many non-transgenic lines by PCR will be time consuming and it costs more. But now there are numerous methods to eliminate the markers in the transgenic plants by marker - free transgenics, selection systems that support the growth of plant in the presence of a particular compound such as sugar, amino acids; co-trasformation strategies, site specific recombination(cre/lox). among these in my point of view site specific recombination is a good one.
Since generation of transgenic plants is laborious and expensive, antibiotic resistance marker genes are used for selection of only transformed cells, by doing so the time and cost is reduced but it is not advisable to do so as it can spread the ABR genes in to the hosts consuming these transgenic plants causing various problems. Why cant we use Reporter genes such as GUS or GFP as selectable marker genes? If we provide specific substrates in the medium which can activate these marker genes that differentiates the transformed and non-transformed cells.
hi I m sorry about this very delayed comment to this topic. I remember in one conference in MSSRF there was a scientist I think ICRISAT who presented his work on marker free transgenics. he generated these plants by just removing the antibiotic marker gene and screening trangenics with PCR, southern and westerns. I personally believe its a great/simple way of generating marker free transgenics. i have myself had few instances where transgenics regenerating from antibiotic enriched medium have been false positives. we will need to confirm the status of these plants using southerns, westerns and PCR so not a having an antibiotic marker shoudl not matter
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