Embryonic Stem Cell Targeting/Blast Injection
The ability to specifically alter the germline of mice using embryonic stem
(ES) cell technology has truly revolutionized modern mammalian biology. In
brief, gene targeting vectors are designed which, through homologous recombination,
replace the wild-type allele of a given gene with a mutated form. The targeted
ES cells are then implanted into day 3.5 blastocysts and transferred to pseudopregnant
mothers. The resulting pups are chimeras between the ES cells (129/SvJ), which
provide a brown coat color, and the C57Bl/6 blastocyst which has a black coat
color. Animals with a high percentage brown coat color are bred and the resultant
pups analyzed for the presence of the targeted allele. The core will electroporate
a single gene targeting construct into ES cells and provide the investigator
with DNA from at least 150 potentially targeted clones. Up to three positive
clones will be injected into blastocysts and chimeric animals will be produced.
Those with the highest percent chimerism will be transferred to the investigators
animal account number and bred by the core. DNA from potentially targeted offspring
will be provided to the investigator. Once positive pups are identified they
will be placed in the animal facility of the investigators choosing.
1) General Considerations
Many types of gene alteration experiments can be performed using embryonic
stem cell technology and as such the targeting vector design will vary from
application to application. In the following example, we illustrate the general
characteristics of a traditional gene-deletion construct which is probably
the most common type of gene alteration vector. For help with other types of
vectors, or for questions related to a particular gene-deletion construct,
please feel free to contact the TMCF office.
Targeting Vector Design
A typical targeting vector is shown below. It contains three basic units consisting
of A) a fragment of DNA homologous to the 5' end of the gene (the 5' arm),
B) a selectable gene marker, most commonly the neomycin resistance gene, and
C) a fragment of DNA homologous to the 3' end of the gene (the 3' arm). In
general, the targeting vector is designed such that some portion of the endogenous
gene is replaced by the neomycin cassette in the targeted allele. Although
many strategies have been employed, we suggest that you delete the initial
coding exon(s) of your gene, thus ensuring that a truncated protein is not
produced.
Typical Targeting Vector
Source of Genomic DNA
Successful gene targeting experiments require optimal sequence identity between
the targeting vector arms and the genomic DNA sequence. Since the ES cells
utilized by the Core are from the murine 129/SvJ strain, it is advisable that
your targeting vector be constructed using genomic DNA from this strain as
well. Genomic screening libraries as well as screening services are commercially
available for the 129/SvJ strain. If this is not possible, please contact our
office before constructing a gene-deletion vector.
Length of the 5' and 3' Arms
Although there does not appear to be any optimal length for these two segments,
longer is usually better. We generally suggest that these segments be between
3-8 kb in length on both sides.
Form and Purity of the DNA for ES cell Targeting
The DNA needs to be purified and prepared according to the standard protocol
provided. Note that it is critical that the DNA be linearized, however it does
not have to be separated from plasmid vector sequences. The concentration of
DNA provided to the core is critical and needs to be ~50 mg total as an ethanol
precipitate.
Scheme for Identifying the Targeted Allele
Devising an appropriate scheme for identifying gene targeting events is often
one of the most difficult aspects of making a gene-alteration vector. It is
strongly advised that you use Southern blotting based on restriction site differences
as illustrated below and not a PCR-based technique. Due to the critical nature
of this experiment and based on personal experience, the TMCF will require
each investigator to design and test a scheme for identification of targeted
clones prior to the initiation of ES cell experiments.
Typical Targeting Analysis Scheme
A) Both the wild type and targeted alleles are depicted. The thick lines in
the targeted allele indicate the 5' and 3' homology regions. Both the 5' and
3' probes are shown. NOTE: At least one of the probes must lie outside of the
homology region or else it is possible to pick up false positives. B) Results
of Southern blotting when the genomic DNA?s are digested with restriction enzyme
R and probed with either the 5? or 3? probe respectively.
2) Instructions for Use of the Gene-Targeting Service
A) Schedule a preliminary appointment with the TMCF director. At this time we can review your experimental design and we can help you with any problems which you may have. This will be a good time to go over targeting vector design and the analysis scheme for targeted alleles.
B) Obtain approval from the animal care and use committee for your project. This is crucial. You cannot have animals without prior approval, therefore the Core cannot begin your experiments until this approval is given. The necessary forms can be attained from the Animal Care and Use Committee (phone # 49163).
C) Obtain approval from the Institutional Biosafety Committee (IBC) for the use of recombinant DNA in your research. Again this must be approved before we can begin your experiments. These forms are available from the IBC office (phone # 41063).
D) Turn in a Gene-Alteration Requisition Form (FORM D).
E) Provide the Core with purified DNA following the protocol found on page 17 of the TMCF manual. Include a copy of the construct along with relevant restriction sites and the location of all probes.
F) Provide a scheme for identification of targeted ES cells, including the results of a control experiment. Again, this is for your protection. It is critical that we be able to identify properly integrated positive clones.
G) Complete a billing form (FORM B). We will give your experiment a project code number. However please give a code name to your experimental construct where we ask for a description of the construct. This will allow you to identify an individual experiment upon receiving your bill. You will be billed for services at the end of the month in which your experiments are initiated, with the actual charge being incurred 2-3 weeks later.
H) Once the gene targeting in the ES cells is complete you will receive genomic DNA from the potentially targeted clones. These will need to be analyzed within two weeks. Once you have identified positive clones complete FORM E and bring it to the TMCF office along with a copy of the experimental results. The positive clones will be thawed and expanded and within two weeks you will receive a second aliquot of DNA from these clones to confirm that all of them are properly targeted. Please repeat the analysis and bring the results to my office. No blast injections will be performed until the clones have been confirmed by a second round of blotting.
I) Individual clones will be injected into blastocysts and you will be informed when litters are born. Highly chimeric pups will be transferred to your animal account number and bred by the Core facility for transmission of the targeted allele. You will be responsible for paying for and ordering the mice for breeding. You may use any strain that you wish, however we recommend that you use C57Bl/6 females. After these pups are weaned you will receive tail DNA for analysis. We strongly recommend that you test the DNA by Southern blotting instead of a PCR based technique. Once heterozygous mutant mice have been identified please fill out FORM F and bring it to the TMCF office along with a copy of the results. These mice will then be transferred to the animal facility of your choice for further mating to homozygosity.
3) Experimental Time Line
Listed below is a time line for the experimental phase of making gene-altered
mice. The time it takes from the planning stage to the initiation of your experiments
will depend in large part on how long it takes for you to obtain all the required
approval forms. Therefore, it is never too early to start this process. The
animal approval process often takes 2-3 months between receiving the forms
from animal care to final approval, so do not wait until you have DNA ready
for us to electroporate into ES cells before you begin this process. Again,
we cannot start your experiments until we have these approvals. This applies
even to the targeting phase of the ES cell work.
4) Forms and Protocols (Adobe Acrobat PDF Files)
Form D. Gene Targeting/Blast Injection Request
Form
Form E. Embryonic Stem Cell Screening
Results
Form F. Gene-Targeted Mouse Screening
Results
DNA Purification Protocol for
ES Cell Targeting
Southern Blotting Protocol