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Guidelines for ES cell electroporation
Introduction:
Gene targeting is the process of disrupting or mutating a specific genetic locus in embryonic
stem (ES) cells, usually with the intention of making knock-out or knock-in mice by injecting
those ES cells into blastocysts.
The targeting construct is a plasmid that contains two long stretches of genomic DNA, called
homology arms, which must match, as closely as possible, the genomic DNA of the ES cell line
being targeted. These arms drive the homologous recombination event that results in insertion of
the construct into the desired locus.
It is the client’s responsibility to design and engineer the targeting construct.
The targeting construct is introduced into ES cells by electroporation; the clones grow under
positive selection by antibiotics and several hundred resistant clones are picked into 96-well
plates. The clones are splitted into duplicate sets, one set is frozen and the other set is used to
isolate DNA for genotyping analysis. The genotyping is usually performed by the client.
The clones that have the correct genotype (heterozygous for the targeted allele) are expanded,
frozen back in multiple vials of each clone, and a second genotyping assay is performed
(Southern blot or PCR) for conformation.
Clones that are positive for both genotyping assays may then be used for injection into
blastocysts to make chimeric mice (see our ES cell injection service).
Ordering:
An ordering form (provided by TMF) is filled up by the investigator and given to the TMF along
with the DNA material. The form has to be entirely completed and the instructions followed
carefully in order to the order to be processed.
It is the client’s responsibility to make sure the targeting construct has been engineered correctly,
mapped, sequenced, and tested.
Turnaround Times:
The minimum time needed to go from electroporation of the targeting construct to clone DNA
ready for genotyping is about 4-5 weeks. Factors that affect this time include growth rates of
different ES cell lines, selection times of different antibiotics, and the number of clones picked.
Southern blotting turnaround times can be quite variable, depending on the status of concurrent
projects and the number of clones picked. The DNA of clones from two 96-well plates can be
digested, electrophoresed, and blotted by one person in one week. Hybridization and
autoradiography require several more days.
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Expansion of positive clones from 96-well plates, freezing back multiple vials, and extracting
more DNA for a second genotyping assay require about 2 weeks.
In order to minimize turnaround times, we recommend optimizing and testing your genotyping
protocol well in advance of the electroporation.
Performance Guarantees:
1 set of electroporation includes 20 millions of cells electroporated, which would give around
200 clones to pick up after selection. In parallel to the construct of interest, a control
electroporation can be performed using a known construct with a known high degree of
recombination (20-30%). Enough clones to cover half of a 96 well plate will be picked from this
control and be genotyped.
In case of failure both of the project and the control, the project will be redone for free.
Service Description:
Electroporation and clone-picking service includes
- linearization and purification of the targeting construct DNA
-
electroporation into ES cells
-
growth of cells under selection
-
picking up to 384 drug-resistant colonies into 96-well plates
-
splitting the plates into 3 sets
-
freezing one set and extracting DNA from the other two
Clone expansion service includes
- thawing from 96-well plates of colonies chosen based on Southern blot results
-
expansion to 24-well and then 6-well plates
-
aliquotting cells and freezing medium into about 8-9 cryovials and storing in liquid
nitrogen
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Mouse Strain Considerations:
Most ES cell lines are derived from one of the many so-called 129 strains of mice. There is a
great deal of genetic variation among these strains (see, for example, Simpson, et al, Nature
Genetics 16:19-27, 1997), so it is important to know exactly which one gave rise to the ES cell
line being targeted. For a list of 129 strains and their most current nomenclature, see the Jackson
Laboratory web page.
If possible, you should always use genomic DNA for the homology arms of your targeting
construct from the same strain of mice the ES cells were derived from, or from the ES cells
themselves. This ensures the best sequence match between the construct and the locus of interest.
Although a small number of mismatches can be tolerated, the frequency of homologous
recombination does go down as the number increases.
See the introduction to our ES cell injection service for a brief discussion of breeding
considerations for chimeric mice made from targeted ES cells.
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