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Now it's time to construct your transgenics and design a cloning protocol to construct your transgenesis vector.
Goals
You will have to identify the suitable restriction sites to clone the cDNA of the gene you are studying inside the viric vector to construct your transgenic mices. At the end of this step, you will have designed your Transgenesis construct.
How to proceed
Download the Transgenesis Lab Notebook to collect your data, and follow the steps:
1) Identify and locate the restriction sites of your cDNA.
- Connect to NEBCutter2.0
- Copy the cDNA sequence of the human gene you want to study. Recover it from the sequences list in Step 2.
- Press "Submit"
- Copy/Paste the sequence of the cDNA on your Transgenesis Lab Notebook and mark the positions of the unique restriction sites of the following enzymes (BamHI, EcoRI, SalI, SmaI, PstI). Identify in the sequence the positions of the ATG and Stop codons to be sure that the restriction sites are not inside the codifying region.
2) Identify and locate these restriction sites on the viric vector.
The whole sequence of the viric vector is not available. You have only an initial map and some complementary informations obteined through gel electrophoresis to estimate the relative position of the rest of the restriction sites.
Download Vector an Gels Document
3) decide which are the restriction sites you will use to insert the cDNA inside the viric vector. Take in account if you want your cDNA to be inserted in sense or antisense orientation.
Key ideas to take into account
- A transgenesis vector is a DNA sequence (usually a modified virus) that can harbor a given DNA sequence (coding for a protein, for example) to insert it in the genome of a host.
- To insert a DNA fragment inside a vector, it is necessary to generate cohesive-edges (cut with the same enzyme) allowing to paste de DNA fragment in the correct sense.
- When a DNA fragment is inserted in its inverted sense (antisense), its overexpression will result in a supression of the expression of the gene in the host by RNA interference.
- Gel electrophoresis allow to study which are the sizes of the digestion of a given DNA with restriction enzimes.
- The sizes of the fragments in simple or double digestions can be useful to locate the relative position of each restriction site.
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