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Mesenchymal stem cells Expressing BDNF

Transfection Stem cells Proteins Protocols

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#1 spikygal

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Posted 09 April 2014 - 08:09 AM

Hello all,

I am a new user here and I am new to my lab, also alone. I have to do make my human mesenchymal stem cells express BDNF a protein that is 25kDa and I have no experience doing it. I have been looking up information on the papers. but the problem is no one writes for a novice in the papers. I dont know how do you get from a cDNA clone that I buy from sinobiological to expressing BDNF in cells. 

If its possible, do you mind outlining the experiments that help me reach my goal as in do RTPCR and then PCR and then..etc..I talked to a molecular biologist who just threw terms eg..vector, ligate, plasmid etc...before I could write them down and research them and then look up the protocols. I need help, please. Thanks so much.



#2 bob1

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Posted 09 April 2014 - 01:12 PM

Ok - if you can get the BDNF clone from a company - this will solve some of your problems.  Make sure that when you get the plasmid that you transform it into some bacteria and keep a glycerol stock or two frozen down.  This will ensure that you have a source of the clone for years to come.    It also pays to sequence the insert on any plasmid coming into the lab just to ensure that it is what the people who sent it to you say it is.

 

Depending on which plasmid the clone is in, you have two choices:

 

1) If it is in a mammalian expression vector (will probably contain a viral promoter such as CMV or SV-40, or perhaps a mammalian promoter), you should be able to just transfect this plasmid into the cells, and this will hopefully give you expression straight off.

 

2) If the BDNF is in a vector used for propagation (e.g. one of the pUC series), then you will need to subclone it into an expression vector before you can transfect and get expression.  There are a number of options here, depending on the vector that you want to insert it into.  The classic way to do this is to identify some restriction sites around your BDNF sequence that are also present on the expression vector (choose non-compatible ends if possible, it makes things easier), but make sure that these sites are also not present inside your BDNF sequence.  You would then digest both the expression vector and the BDNF plasmid with these restriction enzymes and purify the resulting fragments leaving you with linear expression vector and your BDNF clone sequence (usually referred to as an "insert").  Then you would ligate the insert into the expression vector.  Another option is to amplify the BDNF sequence with primers that contain the restriction sites you want, digest the resulting product and the expression vector and ligate.

 

If you don't have the clone then you can extract RNA from cells that do express BDNF, convert this to cDNA, then amplify as above with primers containing restriction sites, digest, ligate.  You could also do topo-TA cloning here, which is faster but much more expensive.

 

Your local university library will very likely have a copy of Sambrook et al., Molecular cloning: a laboratory manual, which will be a very useful resource for you.



#3 spikygal

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Posted 23 April 2014 - 11:20 AM

Thank you so much. The plasmid clone is in probably a mammalian vector.

HG10068-M  Cloning vector ($95) 
 pCMV2-HA ($295) 
 pCMV2-His ($295) 
 pCMV2-Myc ($295) 
 pCMV2-FLAG ($295)

So what am I confused about What should I buy from the choices above: should I buy the cloning vector alone or with the pCMV and which pCMV does it matter?

Please do help and thanks so much for the awesome reply above. 



#4 bob1

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Posted 23 April 2014 - 02:48 PM

Having looked at the product page - the plasmid you will receive it in is a bacterial propagation vector (pMD18-T) and it looks like you will also get an empty one of the other vectors for you to subclone into.  They also have an option for this clone in an expression ready format here, where the sequence is in an untagged pCMV plasmid - this costs $295.  If you buy your first option you will also need something to clone into (you may already have a vector for this - check with your supervisor, and check that it is compatible (compatible restriction sites in the same orientation) with the clone you will receive)

 

For your first option, the vector to choose depends on whether you want the gene to have an added tag, which will be either HA, His, Myc, or FLAG.  His and Myc tags are often used for purification purposes, but HA and FLAG are usually used for immunological methods like western blotting and immunostaining.  FLAG and HA can both be used for purification too, but tend to be a little less specific for these purposes.



#5 spikygal

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Posted 19 May 2014 - 06:38 AM

Thanks so much. I am sorry I am more like an independent post doc with no obvious help. What are compatible restriction sites on a vector for a clone? Thanks I am reading up more on this. BUt sorry theory only gets you so far without any practical work in the field.

Thanks again for the help.



#6 bob1

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Posted 19 May 2014 - 01:51 PM

Each vector (plasmid) should have what is known as a Multiple Cloning Site (often denoted MCS), this will contain a number of restriction enzyme digestion sites in close proximity to each other.  Usually these will be common restriction enzymes like KpnI, BamHI, EcoRI, XbaI, BglII, HindIII etc.
 
Restriction enzyme sites are usually palindromic: e.g., EcoRI has the recognition site G^AATTC (^ shows cut site) on both strands of the DNA (work out the reverse complement and you will see that it is the same as above), which means that it leaves an overhanging end such as:
 

5' G^AATT C 3'
3' C TTAA^G 5'

G            AATTC
CTTAA            G

 
For cloning you need to be able to digest the vector and insert using one or more of these sites, so that the ends are compatible.  For instance you could digest with EcoRI alone and this would give you two ends on the vector that are the same and can be re-ligated (self-ligated) - or they could ligate the insert if both ends of the insert have the same sequence.   However, to make it easier for you and give you a higher chance of getting your vector+insert, not just self-ligated vector, you can use non-compatible restriction sites (different enzymes with different cut sites) at each end of the insert, so that the vector will have a much much lower chance of self-ligation and a higher chance that your insert will be in there.  This also allows you to control which direction the insert is in, which is important if you want expression. e.g. If you wanted to use the EcoRI and BamHI sites on the attached file for insertion of an insert, your plan would look something like:
 

EcoRI  G^AATTC insert sequence G^GATCC BamHI
       CTTAA^G ecneuqes tresni CCTAG^G

 
 As you can see the BamHI and EcoRI do not have the same overhanging end sequences, so they *shouldn't* match up and ligate.
 
The direction is important for expression because the plasmid contains a promoter sequence that allows the RNA polymerase to initiate transcription the DNA, it does this by binding to the promoter and then working its way along until it reaches an initiation codon (ATG) - if your insert is in the wrong orientation, the RNApol won't recognize it and you won't get any protein out.
 
All of the above may not be possible depending on how the company has inserted their product into the vector you are getting, but there are ways around this - you can PCR amplify the insert using primers that contain restriction sites built in, but we can go into that another time

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