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We describe here a detailed protocol for generating gene knockout rats by homologous recombination in embryonic stem (ES) cells. This protocol comprises the following procedures: derivation and expansion of rat ES cells, construction of gene-targeting vectors, generation of gene-targeted rat ES cells and, finally, production of gene-targeted rats. The major differences between this protocol and the classical mouse gene-targeting protocol include ES cell culture methods, drug selection scheme, colony picking and screening strategies. This ES cell–based gene-targeting technique allows sophisticated genetic modifications to be performed in the rat, as many laboratories have been doing in the mouse for the past two decades. Recently we used this protocol to generate Tp53 (also known as p53) gene knockout rats. The entire process requires ~1 year to complete, from derivation of ES cells to generation of knockout rats.
Gene targeting by homologous recombination in embryonic stem (ES) cells is a powerful technique that allows any chosen gene to be genetically modified in a predetermined way1. In the past 20 years, thousands of genes have been modified in mouse ES cells by homologous recombination with gene-targeting vectors, yielding valuable research models. However, this ES cell–based gene-targeting technique was previously only available for the mouse because of the inability to establish authentic ES cells from other species. Recently, we developed a new culture system that can support the efficient derivation and maintenance of germline-competent ES cells not only from mice but also, most importantly, from different strains of rats2–4. This culture system uses a serum-free N2B27 medium supplemented with three small-molecule inhibitors (3i): CHIR99021, PD184352 and SU5402. CHIR99021 specifically inhibits glycogen synthase kinase 3, whereas PD184352 and SU5402 inhibit mitogen-activated protein kinase and fibroblast growth factor receptor tyrosine kinase, respectively2. Subsequently, we found that a more potent mitogen-activated protein kinase inhibitor, PD0325901, can be used to replace both PD184352 and SU5402 (ref. 2). Because the two-inhibitor (CHIR99021 and PD0325901), 2i medium is more effective than 3i medium for deriving and propagating rat ES cells, this protocol describes the use of 2i.
Using the 2i or 3i medium, we and others have established ES cell lines at high efficiency from different strains of rats: dark agouti (DA), Fischer 344 (F344), Sprague-Dawley, Brown Norway, Wistar, Long-Evans and spontaneously hypertensive rats (SHR; refs. 3–9 and unpublished data; Table 1). The availability of robust and germline-competent rat ES cells has opened the door for the application of ES cell–based gene targeting and related genome engineering technologies in rats. The potential utility of this technology was indicated in our recent report on the generation of Tp53 (also known as p53) gene knockout rats by homologous recombination in DA rat ES cells10. Efficient gene targeting by homologous recombination has also been reported in F344 and Sprague-Dawley rat ES cells11. These results have established a strong foundation for the development of gene-targeted rat models and the pursuit of rat functional genomics. The rat offers a complementary model choice to the mouse because rat models have been shown to more closely mimic human disease than mouse models in several areas, including neurodegenerative disease12, nephropathy13, breast cancer14 and rheumatoid arthritis15. Rats are approximately ten times larger than mice, allowing investigators to perform procedures such as nerve recordings, collection of tissue from small structures and serial blood sampling more easily.
Over the years, several technologies have been developed to modify the rat genome. These technologies include pronuclear microinjection16, lentiviral transgenesis17, N-ethyl-N-nitrosourea mutagenesis18,19, transposon mutagenesis20–22 and zinc-finger nuclease–mediated gene targeting23–27. The advantages and disadvantages of each technology are listed in Table 2, and investigators should decide which technology is most appropriate for their applications. Currently, ES cell–based gene targeting is still the most effective technology to generate rats (or mice) with genes modified by homologous recombination and to restrict the genetic modifications to a desired group of tissues or to a chosen period during the development of the animal.
In this protocol, we describe how to generate gene knockout rats by homologous recombination in ES cells. The flowchart in Figure 1 shows the different stages of the process, which requires basic techniques in cell culture, molecular biology, rat embryology and rat surgery. Some procedures, such as karyotyping, vasectomy, embryo isolation and manipulation, microinjection and the transfer of embryos to a pseudopregnant recipient, are provided as a service by animal and transgenic core facilities in many universities and research institutes. Gene-targeting strategies involving homologous recombination have been used routinely to introduce various gene modifications into mice. However, rats are the species of choice in many areas of biomedical research, and we believe that researchers in many laboratories around the world will take advantage of the well-established ES cell–based gene-targeting technology to produce new rat models.
The 2i or 3i medium allows us to establish ES cell lines from different strains of rats at a relatively high efficiency (Table 1). However, using the 2i or 3i medium, we and other researchers could not establish authentic ES cell lines from other species, including rabbits, pigs, cows, sheep, zebrafish and humans. Evidently, new types of ES cell media must be developed to derive ES cells from other species. ES cell lines derived from DA, Sprague-Dawley and Wistar strains of rats have been proven to be germline-competent, but the germline transmission rate is relatively low, and so far the retention of germline competency after gene targeting has only been demonstrated in DA rat ES cells3,4,8,10 (Table 1). Many factors determine the efficiency of gene targeting in rat ES cells and subsequent generation of gene-targeted rats; among these factors are the length of the homology arm, the quality of the rat ES cells and the genetic background of the host embryo chosen for the production of ES cell–rat chimeras. The protocol described here provides a basic platform for optimization of gene-targeting procedures in rats. More information about the limitations of this protocol, as compared with other technologies, is provided in Table 2.
Using the 2i or 3i culture system, we have been able to derive ES cell lines at a relatively high efficiency from all the rat strains that we have tested, including DA, F344, Sprague-Dawley, Brown Norway and Long-Evans (refs. 3,4; and unpublished data). Other groups have reported the derivation of ES cells from other strains of rats5–9 (Table 1). Evidence indicates that ES cells can be established from most, if not all, strains of rats by using the 2i condition. The derivation procedure is relatively simple. After the removal of the zona pellucida with Tyrode’s solution, blastocysts are transferred to a four-well plate precoated with feeders and cultured in 2i medium. ES cell lines can be established from ~50% of the rat blastocysts that had been plated. Rat ES cell derivation efficiency can be increased if the inner cell mass is isolated by immunosurgery after the removal of the zona pellucida (M. Buehr, personal communication). If a particular rat strain is characterized by low efficiency in deriving ES cells, isolation of the inner cell mass will most likely increase the chance of success. Feeders are essential for the maintenance of pluripotent rat ES cells in the 2i condition. Fibroblasts derived from either mouse or rat embryos can be used for rat ES cell culture. Before use as feeders, embryonic fibroblasts must be mitotically inactivated either by γ-irradiation or by mitomycin C treatment28. Rat ES cells grow as loosely attached or floating aggregates in the 2i condition; hence, extra care must be taken to avoid washing away the cells when changing the medium or passaging the cells. For routine passaging, we detach rat ES cells by pipetting and collect them by centrifugation, after which trypsin is added to dissociate the cell aggregates into single cells. Passaging cells in this way can also avoid the carryover of feeders, which adversely affects rat ES cell growth.
Gene targeting by homologous recombination in ES cells has provided a powerful means to elucidate gene function and create gene knockout animal models1. The design of the gene-targeting vector is the first critical step29. Key components of a targeting vector include two homology arms with the same DNA sequences as the genomic DNA fragments flanking the region to be modified, and both a positive and a negative selection marker. Construction of the targeting vectors has been described in detail in various protocols28–32. Although the basic principle for designing a gene-targeting vector is the same for both the mouse and the rat, construction of a gene-targeting vector for the rat requires several modifications to the classic strategy. The first of these is the choice of promoter used to drive the expression of the positive selection marker. In mouse gene-targeting vectors, phosphoglycerate kinase (PGK) promoter is most often used to control the selection marker gene expression. Rat ES cells are very sensitive to drug selection, and the activity of the PGK promoter is too weak for the efficient isolation of drug-resistant colonies of rat ES cells. The cytomegalovirus early enhancer/chicken β-actin (CAG) promoter has a much stronger activity than that of the PGK promoter. The use of the CAG promoter to drive the expression of the positive selection marker has been proven to be very effective for isolating drug-resistant colonies in rat ES cells10.
The second modification to the strategy is the choice of the negative selection marker, which is the gene used to eliminate cells with targeting vectors integrated at non-homologous recombination sites. We use a gene encoding the diphtheria toxin-A chain (referred to herein as DTA) as a negative selection marker, instead of the thymidine kinase gene. Cells with random integration of targeting vectors containing the DTA negative selection gene will produce DTA that kills the cells. As a result, correctly targeted cells can be enriched without the addition of any selection drugs. We found that gancyclovir, at the working concentration of 2.5 μM, is toxic to all the rat ES cell lines that we have tested, including ES cell lines derived from DA, F344 and Sprague-Dawley rats. Gancyclovir also promotes differentiation of rat ES cells maintained in the 2i condition. Consequently, the thymidine kinase gene is not suitable for use as a negative selection marker in the rat gene-targeting vector.
The third modification to the strategy involves the source of homology arms. Currently, there is no information available for DA rat bacterial artificial chromosome end sequence. Homology arms can be amplified from DA rat genomic DNA by using PCR-based methods with high-fidelity DNA polymerases, such as the Expand High Fidelity PLUS PCR System (Roche) or Phusion DNA polymerase (New England Biolabs). The published rat genomic sequences are from the Brown Norway strain of rats33. If there are any sequence differences between the amplified DNA and the published data, it is important to determine whether these differences are polymorphisms specific to DA rat genomic DNA or mutations created by PCR. The homology arms generated by PCR are subcloned into a pUC vector, using Clontech’s infusion method, as illustrated in Figure 2a. The targeting vector is constructed by inserting the 5′ homology arm, the 3′ homology arm and the PGK-DTA-poly(A) cassette into the pCAG-EGFP-IRES-Pac plasmid (Fig. 2b).
Several methods are available for introducing foreign DNA into ES cells, including electroporation, nucleofection and chemical-based transfection. We have evaluated these methods, and our results suggest that electroporation is still the most effective means of generating correctly gene-targeted rat ES cell clones (unpublished data). The variable quality of mouse ES cell lines often hinders the development of the mouse model containing a modified gene locus34. This likely applies to the rat as well. The major factors that affect the quality of ES cells and their ability to generate germline chimeras include culture medium, quality of feeder cells, methods of passaging and the number of passages28. Under suboptimal culture conditions, ES cells progressively acquire chromosomal abnormalities as they are passaged, and this is one of the major reasons as to why ES cells might fail to contribute to the germ-line. The quality of rat ES cells should be regularly monitored by examining their karyotypes, in vitro differentiation potential and expression of pluripotency markers, and also by determining how efficiently they produce germline chimeras, which still remains the gold-standard test3,4,28. Rat ES cells grow as compact dome-shaped colonies in the 2i condition, and as the number of passages increases, some rat ES cells start attaching to the feeders and forming flat colonies. Rat ES cells with flat colony morphology should not be used for gene targeting, because they are most likely karyotypically abnormal10.
Rat ES cells cultured in the 2i condition are very sensitive to drug selection. We administer the selection drug at half of the normal concentration used for mouse ES cells and apply a pulse selection scheme to increase the selection efficiency10. Our tests on ES cells derived from DA, F344 and Sprague-Dawley rats showed this pulse-selection scheme to be highly effective when using puromycin, G418 or hygromycin. However, it is possible that this selection scheme will not work effectively for other drugs or a particular rat ES cell line. If this is the case, the investigators should optimize the selection condition by determining the killing curve of the selection drug on the rat ES cell line that they use. As mentioned above, most of the rat ES cell colonies are either floating or loosely attached to the feeders, and extra care should be taken when changing the medium so that these colonies are not washed away. After drug selection is completed, colonies can be picked using the simple, efficient method depicted in Figure 3. Colonies are detached by pipetting and are pooled together in a sterile tube. Each colony is then placed into one drop (10 μl) of 0.025% (wt/vol) trypsin-EDTA solution to dissociate the ES colony into single cells. In this way, up to 500 colonies can be picked, dissociated and seeded in < 3 h. The dissociated colonies are transferred into duplicate 96-well plates, using a multichannel pipette.
Freezing and thawing of the rat ES cells cultured in the 96-well plate results in a poor recovery rate. To circumvent this problem, we split each rat ES cell colony into two 96-well plates, with one plate having three times more cells than the other. The plate with more cells is used for the initial screening by PCR, which takes 1–2 d to complete, whereas cells in the other plate are maintained in culture. Colonies that test positive in PCR screening are then expanded from the duplicate plate for confirmation by Southern blot, sequencing analysis or both. The strategies for confirmation of the targeting events in rat ES cells by Southern blot are essentially the same as in mouse ES cells28,35. The design of PCR primers and optimization of PCR conditions are critical for the initial screening. One of the PCR primer pair should be located on the genomic DNA beyond the 3′ short arm and the other located on the drug-resistance cassette (Fig. 4). If mouse embryonic fibroblasts (MEFs) are used as feeder layers, BLAST the PCR screening primer sequences against mouse genomic DNA sequences to rule out potential nonspecific amplifications. We recommend constructing a control vector containing the 3′ short homology arm with a 300-bp to 500-bp extension in which one of the PCR primer pair is located (Fig. 4). Rat ES cells transfected with the control plasmid mimic the targeting event and can therefore be used to optimize the PCR screening conditions. The genomic DNA extracted from these rat ES cells is also run in parallel with PCR primers as a positive control for the PCR screening.
Different strains of rats have different suitabilities as models for particular human diseases. For instance, the Long-Evans rat is a good model for diet-induced obesity, and the spontaneous hypertensive rat has been widely used for the study of hypertension. The availability of ES cells from different strains of rats provides investigators the option of producing gene-targeted rat models based on a relevant genetic background. However, for the production of gene-targeted rats by ES cell-based technologies, it is essential that rat ES cells are able to contribute to the formation of chimeras and re-enter the germline. Therefore, before performing gene-targeting experiments on a rat ES cell line, the investigator must determine whether the line is germline-competent and identify the ideal strain combination that leads to its efficient germline transmission.
In the production of ES cell-mouse chimeras, it has been shown that the genetic background of the host embryos is one factor that determines the efficiency with which the ES cell genome is transmitted to the progeny. One of the early findings during the development of gene targeting in 129 mouse ES cells was that the proportion of chimeras that resulted in germline transmission was determined by the genetic background of the host blastocyst. Schwartzberg et al.36 used out-bred MF-1, outbred CD-1 and inbred C57BL/6 mouse blastocysts to produce chimeras with CCE 129 mouse ES cells. All three blastocyst donor mouse strains produced ES cell-mouse chimeras. However, only C57BL/6 host blastocysts produced chimeras that transmitted the genetically modified ES cell genome through the germline. The same result has been observed for the mouse C57BL/6 ES cell lines. The efficiency of germline transmission is increased when the host blastocyst that is used to make ES cell chimeras with C57BL/6 ES cells is derived from C57BL/6-Tyrc-2J/J mice instead of BALB/c, SWR, FVB/N, C57BL/6-Tyrcbrd or 129/Sv mice37–41.
The strain combination is another controlling factor in the germ-line transmission of rat ES cells. Our results suggest that DA rat ES cells can transmit through the germline when they are injected into F344 rat blastocysts, but not when they are injected into Sprague-Dawley rat blastocysts4. ES cells derived from Sprague-Dawley and Wistar rats have also been shown to be germline-competent3,5,8. Another factor to be considered when choosing the strain combination is the coat color genetic background. The identification of chimerism and germline transmission can be facilitated by combining rat strains that have distinct coat colors (Fig. 5). Identification of the ideal strain combination will facilitate the broad application of ES cell–based technology for the production of gene-targeted rat models. In this protocol, we describe gene targeting performed in DA rat ES cells. We have deposited several germline-competent DA rat ES cell lines at the Rat Resource and Research Center in Columbia, Missouri, USA (http://www.rrrc.us/), which has begun distributing the rat ES cell lines to the research community.
Prepare 0.1 M stock solution by diluting 100-μl β-mercaptoethanol with 14.1 ml of distilled H2O. Sterilize through a 0.2-μ filter and store at 4 °C for up to 1 month. ! CAUTION It is flammable; harmful if swallowed; toxic when in contact with skin and eye; and use protective gloves and safety glasses when handling.
Dissolve 5 g in 500 ml of distilled H2O to produce 1% (wt/vol) gelatin stock. Autoclave and store in 50-ml aliquots at 4 °C for up to 3 months.
Add 50 ml of prewarmed 1% (wt/vol) gelatin to 450 ml of PBS. Store at 4 °C for up to 1 month.
Dissolve in sterile 0.01 M HCl overnight at 4 °C to produce a 10 mg ml−1 stock solution. Store in 1-ml aliquots at −20 °C. CRITICAL Insulin does not dissolve readily; hence, ensure that the suspension is mixed well before aliquotting.
Dissolve in sterile distilled H2O to produce a 100 mg ml−1 stock solution. Store in 1-ml aliquots at −20 °C.
Dissolve 3 mg in 5-ml ethanol to produce a 0.6 mg ml−1 stock solution. Sterilize through a 0.2-μm filter. Store in 0.5-ml aliquots at −20 °C. It is good for at least 5 years. Freezing and thawing is fine.
Dissolve 1.6 g in 10-ml distilled H2O to produce a 160 mg ml−1 stock. Sterilize through a 0.2-μm filter. Store in 1-ml aliquots at −20 °C. It is good for at least 5 years. Freezing and thawing is fine.
Dissolve 2.59 mg in 5-ml distilled H2O to produce a 3 mM stock. Sterilize through a 0.2-μm filter. Store in 0.5-ml aliquots at −20 °C. It is good for at least 5 years. Freezing and thawing is fine.
Dissolve 4 mg of CHIR99021 in 860-μl DMSO to produce a 10 mM stock. Store in 100-μl aliquots at −20 °C. It can be stored for at least 3 years.
Dissolve 4 mg of PD0325901 in 830-μl DMSO to produce a 10 mM stock. Store in 50-μl aliquots at −20 °C. It can be stored for at least 3 years.
To 7.187-ml DMEM/F12 medium, add 0.67 ml of 75 mg ml−1 BSA, 33 μl of 0.6 mg ml−1 progesterone solution, 100 μl of 160 mg ml−1 putrescine solution, 10 μl of 3 mM sodium selenite solution, 1 ml of 100 mg ml−1 apo-transferrin and 1 ml of 10 mg ml−1 insulin. Mix well by pipetting and store in 1 ml aliquots at −20 °C. It can be stored for up to 6 months.
To 100 ml of DMEM/F12, add 1 ml of N2 100× stock solution. The final concentration of each component of N2 in the DMEM/F12-N2 medium is as follows: insulin 10 μg ml−1, transferrin 100 μg ml−1, progesterone 20 ng ml−1, putrescine 16 μg ml−1, sodium selenite 30 nM and BSA 50 μg ml−1.
To 100 ml of neurobasal medium, add 2 ml of B27 and 0.5 ml of 200 mM L-glutamine.
Mix DMEM/F12-N2 medium with neurobasal/B27 medium at a ratio of 1:1. To 200 ml of N2B27 medium, add 200 μl of 0.1 M β-mercaptoethanol. The final concentration of β-mercaptoethanol in N2B27 medium is 0.1 mM. Store at 4 °C for up to 1 month.
To 100 ml of N2B27 medium, add 30 μl of 10 mM CHIR99021 and 10 μl of 10 mM PD0325901. The final concentrations of CHIR99021 and PD0325901 in the 2i medium are 3 and 1 μM, respectively. Store at 4 °C for up to 1 month.
Add 5 ml of 2.5% (wt/vol) trypsin, 5 ml of chicken serum and 0.5 ml of 0.5 M EDTA to 500 ml of sterile PBS. Mix well and store in 30 ml aliquots at −20 °C. It can be stored for up to 6 months.
Add 50 ml of heat-inactivated fetal bovine serum, 5 ml of 200 mM L-glutamine solution and 5 ml of penicillin-streptomycin solution to 500 ml of GMEM medium. Store at 4 °C for up to 1 month.
We use standard methods to prepare MEF feeders28. Mitotically inactivate MEFs either by γ-irradiation or mitomycin-C treatment. Plate mitotically inactivated MEFs into gelatin-coated dishes at a density of 2 × 104 to 3 × 104 cells per cm2 and culture in MEF medium. Prepare MEF feeder-coated plates in advance and use within 1 week. MEFs derived from the CF1 mouse strain are used for routine rat ES cell culture. MEFs prepared from other strains of mice should also work for rat ES cell culture. When rat ES cells are under drug selection, they are cultured on drug-resistant MEFs, such as DR4 MEFs, which are resistant to G418, hygromycin, puromycin and 6-thioguanine10.
Dissolve 10% (vol/vol) DMSO in MEF medium. Prepare just before use.
Access the University of California Santa Cruz (UCSC) genome browser website and find the genomic DNA sequence of the gene of interest for performing gene targeting. Retrieve the whole gene sequence and ~15 kb of upstream and ~15 kb of downstream sequences. Import the DNA sequence into sequence analysis software, such as Clone Manager (Scientific & Educational Software). Decide on the location and size of short and long homology arms based on the structure and function of the gene, information of exons and introns, percentages of repetitive sequence and the restriction enzyme sites. The minimal length of the homology arms required for successful gene targeting in rat ES cells has not been determined yet. From our experience, ~6 kb of the long homology arm and ~1.5 kb of the short homology arm are sufficient for gene targeting by homologous recombination in rat ES cells. Design a PCR primer pair to individually amplify the 5′ and 3′ homology arms from genomic DNA extracted from isogenic rat tissues or rat ES cells. The minimum requirements for the PCR primers are described as follows: length, 23–30 bp; annealing temperature, 60–68 °C; and G + C content, 40–60%.
Each primer in this pair contains a 15-bp sequence complementary to the 5′ end of the primer used for amplifying either the 5′ or the 3′ homology arm from genomic DNA. A restriction enzyme site is also introduced into each primer to facilitate the subcloning of the homology arm into the targeting vector backbone plasmid (Box 1 and Fig. 2).
Design a PCR primer pair for the initial screening of correctly targeted rat ES cells. As shown in Figure 4, one of the PCR primer pair should be located on the genomic DNA beyond the 3′ short arm and the other located on the drug resistance cassette. The minimum requirements for the PCR primers are described as follows: length, 20–25 bp; annealing temperature, 58–65 °C; and G + C content, 40–60%.
Mice and rats are housed in the vivarium and maintained under routine husbandry practices, according to the guidelines approved by the Institutional Animal Care and Use Committee. Rats undergoing surgical procedures are housed solitarily for the first 1–2 weeks after surgery. Rat pups are weaned at the age of 4–5 weeks.
The passaging method is, in principle, the same as described in Steps 11–15 of the main procedure, only on a larger scale.
|Reagent||Volume per 50 μl reaction (μl)||Final|
|Paq5000 Reaction buffer (10×)||5||1×|
|dNTP mix (100 mM; 25 mM each dNTP)||0.4||0.8 mM dNTP|
|Forward primer (10 μM)||1||0.2 μM|
|Reverse primer (10 μM)||1||0.2 μM|
|Genomic DNA||100 ng||100 ng|
|Paq5000 DNA Polymerase (5 U μl−1)||0.5||0.05 U μl−1|
|ddH2O||Up to final 50 μl|
|1||95 °C, 2 min||—||—||—|
|2–41||95 °C, 20 s||60 °C, 20 s||72 °C, 1 min||—|
|42||72 °C, 5 min||—|
|Reagent||Volume per 100 μl reaction (μl)||Final|
|Expand PCR buffer (10×)||10||1×|
|MgCl2 (25 mM)||2||0.5 mM|
|dATP (10 mM)||2||0.2 mM|
|dGTP (10 mM)||2||0.2 mM|
|dCTP (10 mM)||2||0.2 mM|
|dTTP (10 mM)||1.7||0.17 mM|
|Dig-dUTP (10 mM)||3||0.3 mM|
|Forward primer (10 μM)||4||0.4 μM|
|Reverse primer (10 μM)||4||0.4 μM|
|DNA template||20 ng||20 ng|
|High-fidelity enzyme (5 U μl−1)||1.5||0.075 U μl−1|
|ddH2O||Up to final 100 μl|
|1||95 °C, 2 min||—||—||—|
|2–41||95 °C, 30 s||60 °C, 30 s||72 °C, 45 s||—|
|42||72 °C, 7 min||—|
Low-efficiency contribution of rat ES cells to the germline is often caused by chromosomal abnormalities in the contributing ES cells. We have frequently observed a high percentage of cells with an abnormal karyotype in gene-targeted rat ES cell clones, despite their parental cell lines having normal karyotypes. Rat ES cells with a normal karyotype can be isolated from these clones by subcloning. This subcloning step may improve the germline competency of gene-targeted rat ES cells.
Troubleshooting advice can be found in Table 3.
Rat ES cell lines can be established from ~50% of the rat blastocysts that are plated under 2i culture conditions. Similar to mouse ES cells, rat ES cells are routinely passaged using trypsin and can be frozen and thawed using conventional methods with typical recovery rates of over 90%. Rat ES cells grow as compact dome-shaped colonies (Fig. 7a) and express pluripotency markers (Fig. 7b–e). Gene-targeted rat ES cells can be routinely generated by homologous recombination. Gene-targeting efficiency in rat ES cells is similar to that in mouse ES cells. Rat ES cells are very sensitive to drug selection and they cannot survive if the selection drug is added continuously, even if they are transfected. To overcome this problem, we add selection drugs at half the concentration used for mouse ES cells and apply a ‘pulsed’ drug selection regimen, as described in the procedure. The majority of the colonies that emerge after applying the ‘pulsed’ drug selection regimen are transfected (Fig. 8). On average, 300–500 drug-resistant colonies can be recovered from 7 × 106 rat ES cells after electroporation with targeting vectors.
The targeted mutations in rat ES cells can transmit through the germline to produce gene-targeted animals10. The quality of rat ES cells is the major factor affecting their germline competency. Therefore, it is important to karyotype rat ES cells before microinjecting them into recipient blastocysts. The genetic background of the host embryo chosen for the production of ES cell–rat chimeras has a dramatic effect on the successful production of chimeric animals that transmit the ES cell haplotype. Germline transmission of the rat ES cell genome can be achieved when DA rat ES cells are injected into F344 rat blastocysts but not when injected into Sprague-Dawley rat blastocysts4,10. The resultant male chimeric rats are crossed with Sprague-Dawley or F344 rats to verify the germline transmission of targeted DA rat ES cells.
We thank N. Wu and Y. Yan for blastocyst injection; G. Chester for ordering rats; R. Montano and colleagues for rat husbandry; and T. Saunders for scientific input. This work was funded by a US National Institutes of Health National Center for Research Resouces grant (R01 RR025881).
AUTHOR CONTRIBUTIONS C.T. and Q.-L.Y. designed the study. C.T., G.H. and P.L. conducted the experiments. G.H. and C.T. wrote a draft of the paper. C.A. and Q.-L.Y. proofread and finalized the paper.
COMPETING FINANCIAL INTERESTS The authors declare competing financial interests (see the HTML version of this article for details).