There are two different types of electroporation services we currently provide:

1) Gene Targeting

2) ES cell-based transgenics (this encompasses siRNA and traditional transgenesis)

 

1) Gene Targeting:

Frequently Asked Questions:

What is Gene Targeting?

     Gene Targeting is the process by which a fragment of genomic DNA is introduced into a mammalian cell line and subsequently locates and undergoes homologous recombination with the endogenous homologous sequence. In our facility we use murine embryonic stem (ES) cells to perform this.

What is needed in my vector to allow Gene Targeting to occur?

     1) Arms of homology: are essential components of gene targeting in ES cells. They are placed in the target vector flanking a positive selectable marker gene and have the same nucleotide sequence as the two segments of the gene of interest with equivalent orientation. To obtain a reasonable targeting frequency the homology of the two arms should be at least 6000-8000 base pairs total. The length of the arms can be uneven, but shorter than 1000bp is NOT recommended for either arm. The ES cell line that we currently use is from a 129SV background; therefore you MUST construct your vector from genomic DNA from this strain of mouse. Small differences in DNA sequence can have very large effects on targeting frequency. You can get 129 genomic libraries from Jeff Conroy at the Microarray/Genomics facility at RPCI.

     2) Selectable Markers :

• positive markers: neo (G418), puro (puromycin), hygro (hygromycin B), hprt (HAT media) **essential**
• negative markers: HSV-tk (FIAU or gancyclovir), dt, diphtheria toxin, hprt **not essential, but helps with enrichment**

     Using both a positive (between the two arms of homology) and negative (outside of the region of homology) selection will enrich for the number of homologous recombinants that will be picked.

What if I want to remove the selectable marker or make a tissue-specific mouse?

Site-specific recombinases can be utilized for this. They catalyze the recombination between two consensus DNA sequences.
The current recombinases are:

• Cre recombinase (flank your element by loxP sites)
• Flp recombinase (flank your element by FRT sites)
• C31 Integrase 3 (flank your element with attPP’ and attBB’ sites)

How would this site-specific recombination occur?

It would occur by either performing another electroporation on your positive ES cells with a cre-expressing or flp-expressing plasmid OR by crossing your positive animal with a mouse transgenic for Cre or Flp driven by a tissue-specific promoter.
To access cre-expressing mouse lines please refer to Andras Nagy’s website: http://www.mshri.on.ca/nagy/

What is the time frame from when I give the transgenic facility my DNA until I have mice?

It depends on what your lab is trying to accomplish and if you want single or multiple electroporations done. See the Gene Targeting timeline for rough estimates.

How will I know if my targeting event occurred?

In order to verify if gene targeting occurred, and in order for us to begin the gene targeting process, you must have developed a proven strategy to determine if gene targeting occurred. You will need a probe that is not contained in the targeting vector that will distinguish the targeted from the wild type allele. Do not use enzymes that contain CpG's as these are frequently methylated in ES cell DNA.
The frozen lines in 96 well plates have a limited life span, which does not allow sufficient time for you to work out your detection strategy after the fact.
If the Southern blots identify targeted lines, those cell lines will be thawed, expanded, and DNA will be prepared for further verification of the targeting event. Because locally duplicated sequences can result from targeted insertion, targeting should be verified with a probe outside the targeting vector on the other side.

Electroporation/Chimera Generation Service Request Form

Gene Targeting timeline

2) ES cell-based transgenics: