See our educational resources below.
Participants will learn to use CRISPR/Cas9 technology and will learn the scientific background needed to make genetically engineered mouse models, rat models or engineered mouse ES cells. You will learn both methods and the principles behind the technology. Research papers providing the experimental background underpinning methods used to engineer genes will be presented and discussed daily. A workshop on using software tools to acquire genomic sequence, Cas9 target selection, and design of DNA donor plasmids is included. You will learn all techniques necessary to produce and identify embryonic stem (ES) cells that have undergone homologous recombination with a targeting vector. Research papers providing the experimental background underpinning methods used to engineer gene targeted mice will be presented and discussed daily. Laboratory training includes daily laboratory sessions on molecular biology techniques such as DNA cloning, DNA sequencing, PCR, testing Cas9 ribonucleoprotein complexes for endonuclease activity, setting up a lab for ES cell culture, media and reagent preparation, normal ES cell culture, preparation of feeder cells, freezing and thawing of ES cells, identification of differentiated and undifferentiated ES cell morphologies, ES cell electroporation, picking ES cell clones, DNA preparation from ES cell clones in 96 well plates, and counting chromosomes in ES cell spreads.
Participants will learn all techniques necessary to produce and identify embryonic stem (ES) cells that have undergone homologous recombination with a targeting vector. Research papers providing the experimental background underpinning methods used to engineer gene targeted mice will be presented and discussed daily. Laboratory training includes setting up a lab for ES cell culture, media and reagent preparation, normal ES cell culture, preparation of feeder cells, freezing and thawing of ES cells, identification of differentiated and undifferentiated ES cell morphologies, ES cell electroporation, picking ES cell clones, DNA preparation from ES cell clones in 96 well plates, and counting chromosomes in ES cell spreads.
Participants will learn all techniques necessary to produce transgenic mice. This includes superovulation, glass microinstrument fabrication, egg collection, identification of pseudopregnant females, surgical transfer of eggs to pseudopregnant recipients, and microinjection of DNA into the pronuclei of fertilized mouse eggs. A majority of trainees produce transgenic mice during training.
Rat transgenic production training is also available.
Participants will learn all techniques necessary to produce embryonic stem cell-mouse chimeras. This includes superovulation, glass microinstrument fabrication, blastocyst collection, identification of pseudopregnant females, surgical transfer of blastocysts to pseudopregnant recipients, and microinjection of ES cells into the blastocoels of C57BL/6 blastocysts. A minority of trainees produce chimeric mice during training.
Participants will learn all techniques necessary to transfer mouse embryos to the reproductive tract of recipients. This includes superovulation, isolation of pathogen free fertilized eggs from infected stocks, transfer pipette fabrication, identification of pseudopregnant females, surgical transfer of eggs to pseudopregnant recipients. All trainees produce offspring from transferred eggs during training.
Consult on design and construction of transgenes and targeting vectors; identification of transgenic founders; and breeding.
This is a brief outline of the steps necessary to obtain transgenic mice or transgenic rats. Simply put, the investigator constructs a transgene with a promoter and a structural gene (for example a reporter gene such as lacZ or a transcription factor). DNA is prepared and microinjected into fertilized mouse eggs. Potentially transgenic mice or rats are born. Transgenic founders are identified, usually by a simple PCR assay, and bred to produce offspring for analysis. Core personnel are available for consultation on all aspects of transgenic research. The transgenic founders are then bred to establish mouse lines and the offspring of each founder are tested for transgene expression. Email [email protected] with any questions.
What is the purpose of your experiment? Do you want to define tissue specific regulatory sequences? Do you want to overexpress a protein in a specific cell lineage? You will need to obtain or clone the desired promoter and structural gene. You will need to establish a gene expression assay. If you expect an observable phenotype in the transgenic mice you should establish methods to measure and quantitate the phenotype. Expression of some genes will be deleterious or incompatible with proper growth and development of the embryo. Special arrangements should be made if you expect embryonic lethality from transgene expression. The expression of a transgene requires that the appropriate transcriptional control elements be included in the DNA construct. A literature search may identify these elements. Preliminary studies in cell cultures are recommended to verify the integrity of the construct and the function of the promoter. However, it is not always possible to predict in advance whether the transgene will have the capability of being expressed in vivo. A review of reporter molecules is available. The Core has a nuclear localized lacZ reporter vector (pnlacf) for investigators who wish to characterize regulatory elements in transgenic mice. A completed materials transfer agreement is required before this plasmid can be distributed to investigators. Commercially available vectors that may be useful in transgenic research include: 1) the CMV-IE promoter for widespread gene expression, 2) tetracycline regulated gene expression systems for inducible gene expression, and 3) luciferase and green fluorescent protein reporter genes.
In transgene design several things should be considered during cloning. For exemple, prokaryotic vector sequences interfere with the expression of some transgenes, thus unique restriction sites at the 5′ and 3′ ends of the construct should be available for vector removal. The transgene should contain unique markers so that its presence can be easily detected in mouse tissue DNA samples and so that its expression can be assayed and distinguished from endogenous gene expression. Sequencing of junction fragments should be carried out in order to confirm that the transgene has a functional promoter, initiation codon, and polyadenylation signal. There are several reports that the inclusion of introns will increase transgene expression (see Review Articles). Under the best circumstances, the transgene is tested for expression in a cell culture system before transgenic mice are made.
We suggest that you establish a PCR assay to rapidly identify transgenic animals. Before you submit your DNA for microinjection into fertilized mouse or rat eggs, the Transgenic Core requires that you provide evidence that you have a PCR or Southern blot assay that detects your transgene when it is spiked into tail DNA at a one copy concentration. A second assay that will detect an endogenous mouse gene, such as beta-globin, or an endogenous rat gene, such as prolactin, is required in order to demonstrate that the DNA preparations are free of PCR inhibitors. Animals are tested with both assays so that no transgenic founder is mistakenly discarded because the tail DNA is contaminated with PCR inhibitors. If your transgene is a bacterial artificial chromosome, it recommended that you use mutliple markers spread along the BAC to make sure an intact copy of the BAC is present in your transgenic founders. You also need to have a Southern blot assay so that you can determine the copy number, integration site number, and transgene integrity in the transgenic founders prior to breeding.
It’s important to show that transgene is expressed. RNA expression can be detected by in situ hybridization or RNAse protection assay with RNA probes. Alternatively, an RT-PCR approach can be used. The protein that is produced must be different from proteins normally expressed in the mouse. There are several ways to achieve this. You may use a reporter gene such as a fluorescent protein that you can visualize directly. You may use a reporter gene that provides for enzymatic amplification of the signal such as beta-galactosidase or human placental alkaline phosphatase. You may use a non-mouse protein such as a human protein or an epitope tagged (i.e. HA, FLAG or myc) protein that can be identified with an antibody that doesn’t bind to mouse proteins. For further discussion of reporters in transgenic mice refer to Saunders, 2003.
We will purify the DNA for you. Simply perform a restriction enzyme digest on your cloning vector to liberate 50 ug of the transgene insert from the cloning vector. Run out a few hundred nanograms of DNA on a minigel to determine whether the digest went to completion and that the bands are the correct size. Bring the remainder of the digest (in a final volume of 100 to 150 microliters) to the Core lab and we will purify the DNA for microinjection from the digest. We use the Nucleospin Extract Kit for purification of microinjection DNA. Please note, if you want use large DNA fragments such as bacterial artificial chromosomes, that there is a specific protocol for the preparation of the BAC DNA for microinjection. Numerous publications show that BACs containing prokaryotic vector sequences are expressed at physiological levels in transgenic mice (Van Keuren et al., 2009). Unlike plasmid based transgenes, removal of vector sequences in not required for BAC transgenes. Transgene constructs are then quantitated and microinjected into (C57BL/6 X SJL)F2 mouse eggs and surgically transferred to recipients. This is provided on a fee for service basis by the Transgenic Facility.. The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your DNA will be added to the microinjection queue in the order that it is received. If you require a different mouse strain (e.g. C57BL/6J or FVB/NTac) we may be able to accommodate your needs. However, there is a surcharge for custom strains since transgenic efficiency is lower in most custom strains.
Tail biopsies from potentially transgenic mice and rats will be obtained 5 weeks after injecting eggs (3 weeks gestation time and 2 weeks of post-natal growth). You will need to extract DNA from the tail tips for use as a PCR template or for Southern blot analysis. The Transgenic Core expects you to test each DNA sample for both the transgene and an endogenous mouse gene or rat gene by PCR. Ideally, you will identify which pups are transgenic before they are weaned at three weeks of age so that only transgenic pups are moved to your animal room. If the testing is not complete then we will transfer all of the pups to your animal room.
Between the time that the transgenic pups are identified and they are 6 weeks old Southern blot analysis should be done to determine how many copies of the transgene integrated, how many chromosomal sites the transgene inserted into, to verify transgenic status and to determine if the transgene is intact. With this information, transgenic founders with a good chance of transmission (at least 5-10 copies) of an intact transgene in a single insertion site can be selected for intensive breeding. Transgenes typically insert in a head-to-tail concatemer. Thus, if you choose a restriction enzyme that cuts once in the transgene you will release DNA fragments the same size as the transgene from multicopy concatemers. The intensity of the hybridization signal will correspond to the copy number of the transgene in the insertion site (see copy standards). Hybridization probes that bind to DNA fragments at the ends of the concatemer will be of unpredictable size since only one of the two restriction sites defining the DNA fragment will be in the transgene. If transgene arrays have integrated onto more than one chromosome the Southern blot will show multiple end fragments corresponding to the number of integration events. The frequency of this occurrence is around 10% of transgenic founders and is usually accompanied by the appearance of a very high copy number on the Southern.
The final stage in the process is to study animals carrying the transgene. Typically, the transgenic founder animals are bred to mice of defined genetic background such as C57BL/6J. Transgenic rats are bred to Sprague-Dawley (Crl:CD (SD)) or other orignating genetic background. Analysis of transgene expression and the consequences of expression is generally conducted in the offspring. The best strategy, if applicable is transgenic founder analysis. This eliminates the time and cost of breeding multiple offspring from each founder.
This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as “knock-out” mice or “gene targeted” mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact [email protected] with any questions. The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.
This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as “knock-out” mice or “gene targeted” mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact [email protected] with any questions. The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.
This is a brief outline of the steps necessary to produce mice with a mutation targeted to a specific gene. These animals are referred to as “knock-out” mice or “gene targeted” mice. The broad outline of the experiment includes: 1) the investigator constructs a gene targeting vector containing a mutation in the target gene; 2) the targeting vector is introduced into ES cells; 3) ES cell clones which undergo homologous recombination with the targeting vector are identified; 4) ES cell-mouse chimeras are produced; 5) the chimeras breed and transmit the chromosome with the targeted gene to their offspring; 6) homozygous animals are produced from the mating of hemizygous chimera offspring; and 7) the phenotype resulting from the genetic mutation is characterized. Core personnel are available for consultation on all aspects of gene targeting research. Contact [email protected] with any questions. The Transgenic Core prioritizes all requests for service on a “first-come, first-serve” basis. Your requests for electroporation, clone expansion, and blastocyst microinjection will be added to the Transgenic Core work queue in the order that they are received.
ES cells are a very sensitive reagent, and most often the weakest link in the process of producing gene targeted mice. Fastidious tissue culture technique is necessary to maintain the pluripotent state of the cells during electroporation, selection, and expansion. In order to maximize successful outcomes, the Transgenic Core maintains ES cells and companion reagents that are quality tested by producing ES cell-mouse chimeras that transmit the ES cell haplotype to offspring. Investigators may choose to use the Core’s Gene Targeting Service (we do all of the tissue culture work, you do all of the molecular biology), or to use space in the Core lab (rent space in our completely equipped laboratory for ES cell culture work), or to do the tissue culture work in their own space. After the cells are electroporated with the targeting vector and put under drug selection, resistant clones are picked and cultured. DNA prepared from the clones is screened and ES cell clones which have undergone homologous recombination with the targeting vector are identified. During screening the clones are cryopreserved in 96 well plates at -80 degrees C. Since these conditions are not optimal for long term storage of cells, it is important to complete the screen in a timely fashion.
After clones are identified, they are brought out of the freeze and expanded. During expansion, additional DNA is prepared and tested, to confirm recombination with the targeting vector, clone morphology is examined, chromosome counts are used to see if the clone is euploid, and a mycoplasma test is done to see if the cells are infected. Investigators may choose to have the Core carry out these tasks, perform them in the Mouse ES Cell Laboratory, or to do them in their own space. In our experience we find that the identification and expansion of five gene targeted clones is enough to generate three euploid, targeted clones for blastocyst microinjection. Once targeted clones have been verified the next step is to…
The Core will microinject ES cells into blastocysts. See the service description for more information. The goal is to produce chimeric animals with high contribution from the ES cell clone and low contribution from the host embryo. This is typically assessed by coat color contribution. ES cells derived from 129 mouse substrains produce agouti fur. When these ES cells are microinjected into C57BL/6 blastocysts (black fur) the resulting mice will appear as agouti on black chimeras. When ES cells are derived from C57BL/6 mouse subtrains the they are injected into albino C57BL/6 mice to produce black on white chimeras. ES cell-mouse chimeras with high coat color contribution from the ES cells are likely to transmit through the germline more quickly than low coat color contribution chimeras. Another sign that the ES cells successfully colonized the host embryo is a distorted male:female sex ratio in the chimeras. Since ES cells are X:Y, a good clone will convert female host embryos to phenotypic males. Thus, the desired outcome is for 75% or more of the chimeras to be male and to include animals that have 90% or more ES cell coat color contribution. Unfortunately, we can not guarantee that all ES cell clones will produce germline chimeras. In our experience we have seen very good clones that produce high contribution chimeras transmit to 100% of their offspring and we have also seen apparently “lethal” clones in which zero animals were born after transferring 90 injected embryos, we have seen “bad” clones in which no animals with ES cell coat color were produced, we have seen “bad” clones which produced low ES cell coat color contribution chimeras. Unfortunately there are no in vitro tests that will differentiate among these outcomes, the only way to discover the germline potential of an ES cell is to inject it. In our experience we find that when three euploid, gene-targeted are used to make ES cell-mouse chimeras that at least one and often two of the clones will produce germline chimeras.
Once the chimeras are produced, breeding is carried out to obtain offspring that carry the targeted gene. Male ES cell-mouse chimeras are mated with C57BL/6 females or albino C57BL/6 mice (also see Breeding Suggestions). The first sign of germline transmission is the appearance of pups with agouti coat color (129 mouse derived ES cell lines) or black coat color (C57BL/6 derived ES cell lines).. Pups produced from sperm derived from ES cells will have agouti coats or black fur. Half of the animals should inherit the targeted gene. Pups that are produced from sperm derived from the C57BL/6 host embryo will have black coats and those from the albino C57BL/6 host embryo will be white. The Transgenic Facility will breed ES cell-mouse chimeras with the appropriate mouse strain for germline transmission on a fee for service basis.Tail biopsies from the pups are screened for the presence of the targeted gene in the same way that you screen for the presence of a conventional transgene.
Once hemizygous mutant mice are identified they are mated to produce homozygous mice. The final stage is to study the animals to determine the consequences of the mutation introduced by homologous recombination in ES cells.
We recommend the either the Sigma-Aldrich GenElute HP Endotoxin Free Plasmid Purification kit or the Qiagen EndoFree Plasmid Maxi kit for the purification of the targeting vector plasmid from bacteria. Please follow the directions in the kit. Electroporation of Qiagen purified DNA has been used successfully by a number of labs. Alternatively, plasmid DNA can be purified by CsCl banding.
Linearize the circular plasmid targeting vector DNA with a restriction enzyme that cuts once in the plasmid vector backbone, not in the arms of homology that will mediate recombination. Digest a sufficient amount of plasmid DNA (~300 ug) to produce 200 ug for ES cell electroporation. Run a DNA on a minigel to verify that digestion is complete. Extract the DNA with 1 volume of phenol-chloroform, then with 1 volume of chloroform and precipitate by adding 0.1 volumes NaCl and 2 volumes cold ethanol. Resuspend the DNA in sterile TE (10 mM Tris-HCl, pH 8.0, 1.0 mM EDTA) at 1 mg/ml. Prior to electroporation, the Transgenic Core will verify the concentration and run it on a minigel to check the size.
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