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UNC Animal Models Core Facility Gene-Targeting Services |
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Information
for Clients
General
Information
The UNC Animal Models Core Facility was established to provide mouse genetic modification services, reagents and related technologies. Services for creating transgenic mice include isolation of the transgene fragment and pronuclear microinjections. Animals are typically a C57/Bl6 X C3H hybrid strain, but can be made in a specific strain if needed for an additional cost, which varies depending on the cost of the strain. Gene targeting services include assistance with the design of targeting vectors, creating gene-targeted ES cells and blastocyst microinjections of the ES cells to create tarnsmitting chimeras. Related services include creating homozygous ES cells from targeted ES cells, Cre- or Flp-mediated excision in ES cells, karyotyping, tail DNA purification, PCR screening, Southern blotting, embryo/ovary/sperm cryopreservation, ovary transplants and in vitro fertilization. Reagents available include mouse genomic DNA (for PCR and Southerns), a mouse genomic library (strain 129/SvEv in l phage) and several plasmids useful for engineering targeting vectors. To obtain services, clients must fill out a service "Gene-Targeted ES Cell/Mouse Request and Agreement" form. This form assures the facility that an "Application to Use Live Vertebrate Animals" covering the genetically modified mice has been approved by the Institutional Animal Care and Use Committee (IACUC). It also identifies the grant or source of funds to pay for reagents and services, a contact person in the client's lab and acts as a contract between the facility and client. Copies of the forms and a price list are attached. A description of the specific services available, the responsibilities for the client, the reagents needed from the client and limitations of the services is attached, as is a list of protocols available.
Gene-Targeting Services
The UNC Animal Models Core Facility will create gene-targeted ES cells from targeting DNA vectors supplied by the client and will generate gene-targeted mice from the targeted ES cells.
1. Targeting Vector Design:
General Considerations and Client/Facility Responsibilities
Clients should consult with the facility director prior to beginning construction of their targeting vector and positive control plasmid. There are several plasmid backbones available from the facility that are designed to accept the arms of homology and provide for positive-negative selection. One plasmid (OSdupdel, made and kindly provided by Dr. Oliver Smithies) has the neomycin-resistance gene (neo) flanked by loxP sites so that neo can be easily removed by Cre treatment. In addition, it is designed so that the same arms of homology can be used to make gene duplications as well as deletions. The "short" arm of homology should be between 1 and 2 kb long. A positive control plasmid for PCR screening would contain the entire a fragment of genomic encompassing the short arm of homology, but slightly longer, to allow for a targeted locus-specific set of primers to be used in the screening of ES cells. The long arm can be up to 10 kb long, but a length of 5-10 kb is acceptable for a reasonable frequency of targeting at most loci. The source of the arms of homology should be considered in constructing a targeting vector. In some instances, isogenic DNA may be required. The facility uses strain 129/SvEv ES cells and a phage library of this strain is available to clients. However, the facility will not screen the library for a client. Other 129 strains have, in general, yeilded acceptable targeting frequencies, with the exception of strain 129/SvJ. Arms of homology derived from this strain have yielded very low frequencies in non-isogenic ES cells and may not acceptable. The facility gives no guarantee for targeting experiments using SvJ-derived arms of homology and clients wishing to pursue targeting using 129/SvJ-derived arms are responsible for the payment of ES cell targeting charges following the screening of a reasonable number of ES cell clones (400). The deletion size should also be considered. Deletions of 10 kb or less are easily obtained following positive-negative selection. Deletions above 20 kb can be extremely rare and may require several attempts. It is not necessary to delete an entire gene to produce a knockout. For large genes, there are three possible ways to affect gene inactivation: 1) Gene Disruption- use homologous recombination to insert the neo gene in a critical exon. This works quite well when a critical exon is known. The neo gene is typically inserted into the exon, in an orientation opposite to that of the targeted gene and the exon is all or partially deleted in the process. 2) Partial Gene Deletion- it is possible to eliminate the function of a gene product by deleting a portion of the gene instead of the entire gene. Usually, a deletion removing the promoter and/or several critical exons is equivalent to deleting the entire gene. 3) Cre-Mediated Deletions- it is possible to make very large deletions using Cre-lox technology, but this requires two targeting events and treatment of the double-targeted ES cells with a Cre-expressing plasmid. In this case, the client would be charged for two ES targeting experiments and a Cre treatment.
2. Submitting Targeting DNA
Targeting DNA should be supplied to the facility after it has been linearized with a restriction endonuclease. In most cases, the cut DNA does not need to be purified. Instead, the endonuclease can be heat-inactivated (treatment at 70oC for 20 minutes). Consult with the endonuclease supplier for information on heat-inactivation. Those that cannot be heat-inactivated should be extracted with 500 ml of chloroform-isoamyl alcohol (24:1 mixture), ethanol precipitated and re-suspended in sterile TE (20 mM Tris/0.1 mM EDTA, pH 7.5). The client should provide a minimum of 50 mg of targeting DNA at a concentration not less than 200 ng/ml. At the time of submission, the client must submit a completed "Gene Targeting Request" form along with a photograph of an agarose gel showing 1) the DNA before cutting, 2) the DNA after cutting and 3) an appropriate molecular weight marker. A detailed map of the vector is also required. In addition, the client must demonstrate the ability to detect the targeted locus by PCR and/or Southern hybridization assays. If the primarty ES cells screen is to be by PCR (performed by the facility), the client must supply the facility with a positive-control plasmid, as well as the primers that distinguish the targeted allele from the wild-type allele. Since the facility primarily uses the Qiagen PCR Core Kit (Cat. # 210223), including Q-solution, it is advisable that the client establish a PCR protocol with this system. Should another PCR system be required, the facility may request that the client provide the necessary reagents to the facility or perform the assay in their own laboratory. If the primary assay is to be by Southern hybridization (performed by the client), the client must demonstrate the ability to detect ther wild-type allele using at least 2 different restriction digests of ES cell DNA (available from the facility). A clear plan for analyses of potential recombinants must be presented to the facility director for approval of the project. Gene targeting experiments will not be initiated until the client has presented documented ability to detect the targeted locus. ES cells testing positive for the targheted locus by PCR must be confirmed by a Southern hybridizatiuon assay. It is for this reason that the client should establish both PCRT and Southerns assays priuor to initiating a gene targeting experiment. The facility is obligated to screen at least 200 ES colonies by PCR or to provide the client with a minimum of 80 colonies to screen by Southern hybridization.. In signing the "Gene-Targeted ES Cell/Mouse Request and Agreement" form., the client accepts these conditions and agrees to pay for services whether or not a targeted ES cell line has been obtained. If recombinant ES cells have not been obtained following 2 rounds of electroporation , selection and screening, it is advisable to meet with the facility director to discuss options. Every effort will be made by the facility to make the cells needed by the client.
3. ES Cell Targeting
Prior to the commencement of gene targeting in ES cells, the client must demonstrate an ability to detect the targeted locus by PCR (preferable) and/or Southern hybridization. Because ES cells must be grown in higher quantities for Southerns (requiring more passages, media, technical time, etc.) fewer ES clones can be provided for screening by Southerns than for PCR screening and the cost to the client is higher. For these reasons, it is strongly recommended that the client develop a PCR procedure for primary screening of the ES cells. This allows very low passage cells to be expanded and frozen, increasing the probability of transmission. To develop the PCR screen, 10 fg of the positive control plasmid is mixed with 5 microliters of ES lysate in the PCR reaction. ES lysate is provided by the facility. Since the PCR primary screen will be performed by the facility, it is preferable that a simple PCR protocol be established. The facility uses the Qiagen PCR Core Kit for most applications. If special conditions or a specific Taq polymerase must be used, the client may be required to provide reagents to the facility, or to perform the PCR screen themselves. For each electroporation, the facility picks and PCR-screens approximately 150 colonies. If a targeted cell line is not detected, the facility repeats the procedure up to two times more. If targeted cells have not been produced, the client should meet with the facility director to review the construct design and discuss options. The client is not liable for negative results due to the failures on the part of the facility (such as clone loss due to incubator malfunction). However, clients are responsible for paying for negative experiments due to errors on their part (such as providing the wrong targeting DNA, wrong PCR primers, etc. to the facility). PCR-positive clones are expanded, frozen in liquid nitrogen and grown in large quantities for Southern hybridization to confirm correctly targeted ES clones. The facility will provide purified DNA to the client for this purpose. The frequency of PCR-positive clones being correctly targeted typically ranges from 50 to 100%. In cases where a PCR screen for targeted ES cells is not possible, ES colonies must be screened by Southern hybridization. The client must demonstrate the ability to detect single bands with at least two different restriction enzyme digests of strain 129/SvEv genomic DNA (provided by the facility). A map of the locus showing predicatble band size differences between the wild-type locus and the targeted locus and a copy of the Southern results must be provided to the facility for the records of the project. A clear photocopy of the results is acceptable, but the original film or phosphoimage scan must be presented to the facility to verify acceptable results. The facility will supply the client with ES cells from approximately 48 colonies per electroporation for Southern screening. A protocol for isolating the DNA will be provided. If the client cannot perform the Southern screen, this service is available from the facility for an additional charge. If a positive clone is not identified in the first round, the facility will repeat the electroporations up to two times and supply the client with another 96 (approximate total) colonies for screening. It is recommended that the client meet with the facility director if at least one positive clone has not been found in the first three attempts.
4. Generating Transmitting
Chimeras and Homozygous Null Animals
Before any animal work can begin, including blastocyst
microinjections of the targeted ES cells, the client must provide the
facility with the IACUC number of an approved Application to Use Live
Vertebrate Animals. This number assures the facility that the proposed
experiments have been approved and that proper animal handling and care
protocols are in place. For UNC clients, this number will be used to
obtain cage cards that will be placed on the cages of mice used for
generating the chimeras for the client. Gene-targeted mice are produced by
microinjection of the gene-targeted ES cells into strain C57/Bl6 mouse
blastocysts. The ES cells can either be those made by the client or those
made by the facility. Those made by the client must be expanded and stored
in liquid nitrogen in the facility and this will incur an additional
charge of $500 per cell line. The cells also must be karyotyped and
screened for mycoplasma contamination before microinjection. There is no
guarantee on the production of chimeras or transmission from targeted ES
cells made outside the facility. Targeted ES cells made within the
facility are expanded for injection without an additional charge and carry
a qualified guarantee of at least 4 high (> 75%) chimera males, or
transmission of the intended genetic modification, whichever comes first.
At approximately 3 weeks of age (weaning) all chimeras are transferred
into the care of the client. Animal space must be arranged by the client
within the institution to receive the animals. At UNC, animal space can be
obtained only by contacting Dr.
5. Suggestions for Initial
Breeding of Chimeras.
Embryo donors used to make chimeras are from strain C57Bl/6 (commonly called B6), which have a black coat. The ES cells used typically are derived from strain 129 and are either from white coated or brown (agouti) coated animals. When the ES cells are implanted into B6 embryos, some of the ES cells migrate to the genital ridge and become germ cells. Because germ cells are derived from the same cell lineage that gives rise to the skin (primitive ectoderm), the probability of germ cells originating from the implanted ES cells can be roughly equated with the degree of chimerism. In other words, a chimera with a high proportion of white or brown fur has a high probability of transmitting the targeted gene to its offspring. High chimera females are rarely produced, but may also transmit the intended genetic modification and should be mated. As a rule of thumb, male chimeras below 40% and female chimeras below 80% rarely transmit. If space is not an issue, all chimeras should be mated to wild-type animals. If space is an issue, then only those males above 40% and females above 80% should be mated. When the 129 genome is passed to the offspring (typically, chimeras are crossed with a B6 animal in the initial matings) pups with non-black coats are produced. These are loosely referred to as agouti's, but this is a misnomer for ES cells that are not derived from a mouse carrying the agouti locus. However, a brown, or other non-black coat color in the pups is an indicator that the ES cells had successfully populated the germ cells of the chimera. Because the ES cells are heterozygous for the engineered genetic modification, the germ cells dserived from the ES cells can be either wild-type of mutant. Thus, 50% of the "agouti" pups will carry the mutant allele. Agouti's with the targeted allele (+/-) should be identified by PCR and/or Southern hybridization and mated with each other to produce wild-type (+/+), heterozygous (+/-) and homozygous (-/-) animals for initial phenotyping. Altenatively, agouitis can be mated with wild-type animals (such as B6) to produce first back-crossed heterozygous animals that can be interbred to produce null animals for phenotyping. The choice of either route may be dictated by the number and sexes of the agoutis produced. For example, if only male agoutis are produced, the former option would not be possible.
6. Extended
Breeding Options
There are several breeding options that can be adopted. The option chosen may be critical, as there are a number of phenotypic differences among the various strains of mice used for genetic research and it behooves the client to become familiar with strain differences as they relate to their gene and expected phenotype (s). A. Establishing an Isogenic Strain. If there are no known differences in the gene or expected phenotype, the best option is to create an isogenic mouse line to avoid potential influences that a mixed genotype might have on the phenotype. The only way to generate an isogenic mouse line is to mate a transmitting chimera with a strain identical to that from which the ES cells used to make the chimera were derived (for example, strains 129 SvEv). A common approach is to first mate the chimeras to strain B6 mates to determine which of the chimeras are transmitting the 129 genome. However, when crossed to an isogenic animal, coat color cannot be used to identify germline transmission and all of the offspring originating from the chimeras must be genotyped to detyermine transmission of the targeted allele. This is where establishing a genotyping protocol by PCR becomes very important in reducing the time and costs of experiments. Heterozygotes can then be interbred to produce genetically uniform animals that vary among themselves only at the targeted locus. B. Establishing a Congenic Strain. When strain differences are suspected to influence the phenotype of the gene-targeted mouse, it is advisable to reduce strain-related variability by establishing a congenic mouse strain. This is accomplished through multiple back-crosses onto a genetic background that is favorable to the phenotype. Back-crossing is achieved by mating the chimera or its agouti (+/-) offspring with the strain of animal needed to support the phenotype (for example, strain Balb/C). The offspring from this mating are considered back-cross 1 or N1. In the example, heterozygous N1 offspring would then be mated with wild-type Balb/C animals to produce back-cross 2 (N2) offspring. Again, heterozygous pups would be mated with wild-type Balb/C animals to produce N3 offspring. The cycle of selecting heterozygous animals and mating these with wild-type animals continues until back-cross 9 (N9) is achieved, at which time a "negligible" amount of the 129 geneome remains associated with the modified allele and the strain is considered congenic and genetically homogeneous. It is well in the interest of the client to begin back-crossing their mutant mouse strain as soon as possible. If an interesting phenotype is found in their animal, but its penetrance is less than 100% in the first group of homozygous animals produced for the study (typically mixed genotypes), it is not unusual for a reviewer to request that the experiments be repeated in a congenic strain. Since back-crossing to N9 can take several years and there are likely to be competitors making similar animals, a late start in back-crossing can make the difference between a high profile and a low profile publication. It is possible to develop a congenic strain more rapidly through the use of strain-specific markers (speed congenics) to identify back-cross heterozygous offspring that carry a high proportion of DNA from of the strain needed (e.g., Balb/C). In this way, a congenic strain can be produced in approximately 6 back-crosses. Charles River Laboratories provides this service, but is expensive.
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