Build a complex genomic locus sequence using CLC Main

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Use the CLC main workbench and Invitrogen vector sequences to reconstitute a Flp recombinase generated locus present in Flip-In cells

Introduction

The Flp-In System allows integration and expression of your gene of interest in mammalian cells at a specific genomic location. The Flp-In System involves introduction of a Flp Recombination Target (FRT) site into the genome of the mammalian cell line of choice. An expression vector containing your gene of interest is then integrated into the genome via Flp recombinase- mediated DNA recombination at the FRT site (O'Gorman et al., 1991[1]).


trex_integration.png
References: [2][3]

Why constructing your genome locus region?

Obviously, having the in-silico copy of your own, freshly integrated, Flip-In construct can be very handy if you need to quantify transgene expression using RNA-Seq or plan any experiment related to the exact sequence built in your Flip-In cells. The CLC Main workbench application allows manual cloning of the different vectors involved in the process. This manual cloning is a relatively straightforward process but it requires CLC manipulations that may seem complex to the average user (as was the case for me).

We present here a full workflow, starting from available vectors and ending with the full sequence of the T-Rex locus, as expected in your cells. You can download the vectors used in this tutorial from the link at the bottom of the page in CLC format. Import the data directly from the zip file while in CLC and you wil obtain the sequences required to reprocude the tutorial. If you do not work with T-Rex but instead with the constiotutive Flip-In system, please follow the same procedure starting from the pcDNA?-FRT vector of your choice.

More information

Handicon.png The following tutorial was prepared with help from the CLC support team (thanks Camilla!)

More information on manual cloning can be read in the CLC help pages [4]:

Frequently used mouse right-click features

manual_cloning.png

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Vectors and Rationale

After importing the provided zip file, you should have the following two vectors in your CLC file manager.


01-start_vectors.png

The pFRT-LacZeo vector was inserted as a single copy into the genome of your Flip-In cells at a earlier stage (in your lab or at Invitrogen), the pcDNA5-FRT (TO!) vector is used to clone your gene of interest (GOI) and recombinate it in the inserted locus with help of the transciently expressed Flp enzyme (from pOG44). Another vector of interest is the pcDNA6_TR plasmid that expressed constitutively the tetracycline repressor required for the Tet-ON system (T-Rex). Please ignore other CLC imported file at this point.

The integrated vector expresses LacZ-Zeo under the control of the SV40 promoter; The ATG of the fusion is directly followed by a FRT site.


pFRTlacZeo.png

The cloning vector also includes a FRT site followed by the Hygromycin resistance gene but lacking a promoter and a start codon. It cannot therefore be expressed (not transcribed nor translated) if intergrated randomly in the genome. It also contains the tetR-repressible CMV.P-cassette in 5' (with 'MCS' for the muclti-cloning site region) where the gene of interest is cloned by Topo cloning or by the conventional use of endonucleases and ligase.


pcDNA5_FRT_TO_selection.png

The next sequence is that of the Tet-R vector for those who will apply this tutorial to the T-Rex system. This vector was generally stably transfected in the cells prior to Flip-In recombination and is not located in the same locus as the transgene target.


pcDNA6_TR_selection.png

Upon homologous recombination by the Flp enzyme, the full pcDNA5-FRT-TO construct will be integrated at the FRT site of the pFRTlacZeo locus; leading to a switch in the resistance from the ZeocinR to HygromycinR, and to the integration of the tetR-repressible expression cassette (see figure above, point 3.).

Manual Cloning Tutorial

We now proceed with the manual assembly of the two vectors to generate the fully recombined locus.

start the workflow

  • The CLC tool used for this is the cloning tool
02-cloning_tool.png

 

  • Upon starting the tool, locate the two vectors and add them to the right selection window
03-select_vectors.png

 

  • Open the next window
04-open_results.png

 

  • Review the vector list from the top pop-down menu (note that both vectors are circular)
05-two_circular_vectors.png

 

Prepare the recipient vector

  • Select the 'recipient' locus sequence (pFRTLacZeo)
06-zoom_to_FRT_in_LINEAR_recipient-vector.png

 

  • Zoom into the sequence to locate the FRT region, then select the right half of the FRT site, starting one base left of the Xba1 site (this is the position where Flp recombines the two FRT motifs)
07-select_FRT_right-half.png

 

  • Right-click on the selected stretch and cut the vector directly left from the selection using the menu item
08-cut_before_selection.png

 

  • Change the check-box to show the sequence as linear
09-review_LINEAR_results.png

 

Prepare the donor vector

  • select the other vector from the top menu
11-zoom_to_FRT.png

 

  • Zoom into the sequence and locate the FRT site then select the left-half similarly to above
12-select_left-half_of_FRT.png

 

  • This time cut right from the selection using the right-click menu
13-cut_right_from_selection.png

 

assemble the two vectors

  • Change back to the linearized recipient vector from the top menu and right-click on the label (sequence name on the left) to insert a fragment into it
14-right-click_on_sequence-name_insert-another-sequence.png

 

  • Choose the linearized donor vector as insert
15-select_donnor_vector.png

 

  • Review the results to check for errors and validate your choice with 'Finish'
16-review_and_finish.png

 

Technical.png At this point, the construction work is done but the results are not yet saved to the disk.

Cleanup and polishing steps

  • Right-click the construct label (name) and make the construct circular in order to perform the next steps
17-click-name_and_circularize.png

 

  • Right-click again on the label (name) of the construct and ask CLC to save it as a new file
18-click_name_and_open_sequence.png

 

  • Pick good name for your file and save it
19-save_to_new_file.png

 

  • Global view on the recombined locus. Note that each FRT site has been split in two half by the cloning-process, you may wish to merge them back to two full FRT sites
20-improve_display_clean_features.png

 

Rotate the vector

The construct is now starting at the FRT site which is not very useful since we are more interested in the GOI cassette.

It is a good idea, at this step, to rotate the vector to start at (for instance) the non-unique Pst1 site present between the noATG_LacZeo and the SV40 promoter driving the expression of the new Hygro resistance (round 8000bps). By doing this we reproduce what Invitrogen shows in their manual and is likely integrated in the cells. It will also put your expression cassette in the middle of the locus and isolate it (better) from possible external influences like Trans-Effects from neighbor genes thanks to the different polyA signals. Note that the coordinate system is finally adjusted to start at +1 just after the CMV promoter.

  • After rotating and 'making the vector linear (right-click on its name as usual)
20b-improve_display_clean_features.png

 

  • Zooming into the cassette to show the MCS between the de-repressible CMV promoter and the polyA signal
21-GOI-cassette_here-empty.png

 

  • Finally, zooming in the downstream region of the recombined HygroR transgene which has inherited from the SV40 promoter and ATG of the late LacZ-Zeo transgene (itself inactivated in the process). The ATG-codon and Hygro-sequence are now in-frame and expressed as a single polypeptide
22-ATG-FRT_and_Hygro_are_now_in-frame.png

 

The final full locus should look like this

Flip-IN_locus_details.png

download tutorial files

Download exercise files here.

Use the right click of your mouse to download the CLC-archive without unzipping it.
CLC_archive.png

References:
  1. S O'Gorman, D T Fox, G M Wahl
    Recombinase-mediated gene activation and site-specific integration in mammalian cells.
    Science: 1991, 251(4999);1351-5
    [PubMed:1900642] ##WORLDCAT## (P p)

  2. http://tools.lifetechnologies.com/content/sfs/manuals/flpinsystem_man.pdf
  3. http://tools.lifetechnologies.com/content/sfs/manuals/flpintrexcells_man.pdf
  4. http://www.clcsupport.com/clcmainworkbench/current/index.php?manual=Introduction_CLC_Main_Workbench.html



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