Gene Trap How-To

How Do I:

Learn more about what gene trapping is
Learn more about the different gene trap vectors CMHD uses
Learn how CMHD generates gene trap clones in a high throughput manner
Take care of ES cells
Confirm my clone once I receive it
Identify where the gene trap vector has inserted for genotyping purposes
Isolate DNA from the ES cells and mice
Design a PCR genotyping protocol
Increase my odds of germ line transmission

Learn more about what gene trapping is:

View Gene Trap Primer

View Gene Trap Vectors

Gene Identification of Gene Trap Insertion Using 3' RACE

Learn how CMHD generates gene trap clones in a high throughput manner

Identify where the gene trap vector has inserted for genotyping purposes

Identification of Insertion Site Using Genomic DNA

As soon as you have expanded your ES cells, DNA can be isolated and the determination of your insertion site can be done. This is very important for genotyping your mice.

Technique 1 Long range PCR

You need to determine where within the intron the vector has inserted in order to determine a PCR genotyping protocol. Long range PCR is a good first step if your intron region is not too large. Using gene specific primers (GSP, in black) at the ends of the flanking exons, in conjunction with the vector specific primers, you can use any commercially available kit to perform long range PCR. Resulting bands are sent for sequencing to identify flanking intronic sequence. Using this method we have amplified up to 18kb to map the insertion.

If long range PCR is unsuccessful, primers can be designed throughout the intron (in red) and used in conjunction with vector primers to try and pull up a band using PCR. Resulting bands are sent for sequencing to identify flanking intronic sequence

Isolate DNA from the ES cells and mice

View Tail DNA Isolation

Design a PCR genotyping protocol

Developing a PCR Strategy for Genotyping Your Mice

Once you know your insertion site, a PCR genotyping protocol can be optimized. This is essential to maintaining your mouse strain and it is advised that you start working on this in advance/conjunction of making your mice. An example is shown below using 2 primers designed within the intron of the gene (ISP), flanking the vector insertion site, and 1 vector specific primer.

Increase my odds of germ line transmission

http://www.millipore.com/catalogue/item/scm001