Conditional Gene Targeting

As presented in Subheading 3.2., conventional gene targeting generates a modified allele in all cells of the mouse. Conditional gene targeting refers to a gene modification in the mouse that is restricted to certain cell types (tissue-specific), to a particular stage within development (temporal-specific), or both (11). This methodology is particularly useful for elucidating a complete picture of gene function in cases where: (1) the conventional knockout leads to an early lethal phenotype, and/or (2) genes may exert their function at several stages of ontogeny and in different cell types.

For a conditional gene-targeting experiment, two separate mouse strains are typically generated and intercrossed; one carrying the conditional allele, a gene segment (or the entire gene) flanked by two loxP sites, and another expressing Cre recombinase (see Subheading 3.2.2.) in certain tissues or cell types, either constitutively or following induction by exogenous stimuli (Fig. 6A). In offspring, only cells expressing the Cre delete the target gene segment. This binary design offers great versatility to the system, as different Cre transgenics can be crossed to the same conditional allele to study gene function in a variety of cells.

3.3.1. Engineering Conditionally Targeted Alleles

Gene constructs that can be modified by Cre are usually introduced into the mouse genome by gene targeting (see Subheading 3.2.). Therefore, the rules of conventional gene targeting also apply in this case. The aim of gene targeting for the production of Cre-modifiable lines is to introduce loxP sites at specific and predefined places into the gene of interest, so that Cre-mediated recombination between these loxP sites will usually result in reactivation, inactivation, or replacement of a gene. All three types of conditional gene modifications have been successfully applied in mice (41-44).

Currently, the most popular use of conditional genetics is conditional gene inactivation using the "three-loxP" strategy developed by H. Gu and colleagues (41). The targeting vector should contain three loxP sites all in the same orientation, two flanking the selectable marker and the third one placed at such a distance from the loxP-flanked selectable marker so that the part of the gene to be modified is included between them (Fig. 6B) (see Note 23). After targeting of the 3-loxP vector into the genomic locus, the selectable marker can be removed by transient expression of Cre in fertilized oocytes as described in Subheading 3.2.2. As all three loxP sites are in the same orientation in the targeted allele, there will be three possible recombination products due to partial excision by Cre. After selection of the desired conditional allele among the

Fig. 6. Conditional gene targeting requires crossing between two separate mouse strains, one carrying the conditional allele and another expressing tissue-specific Cre recombinase (A). In the double mutant mouse, if Cre is expressed in a cell it removes the loxP-flanked region rendering an allele either inactive (B) or active (C). Examples of such applications are illustrated for the TNF locus.

Fig. 6. Conditional gene targeting requires crossing between two separate mouse strains, one carrying the conditional allele and another expressing tissue-specific Cre recombinase (A). In the double mutant mouse, if Cre is expressed in a cell it removes the loxP-flanked region rendering an allele either inactive (B) or active (C). Examples of such applications are illustrated for the TNF locus.

null recombination products, and generation of the conditional mice, a further crossing to various strains expressing Cre recombinase in a cell-type-specific or inducible manner is required for conditional inactivation of the gene. The target gene becomes inactivated only in cells expressing Cre, but remains active in all other cells of the body.

For conditional gene reactivation, the gene is initially inactivated by the introduction of loxP-flanked inhibitory sequences into an area important for gene expression. Such areas may be a specific intron or a site between the transcription initiation and the ATG starting codon of the gene of interest. Removal of the inhibitory sequences by Cre-mediated recombination should result in a fully functional gene. By crossing to a Cre-expressing line, the inhibitory sequences are removed only in those tissues, or only after those developmental stages, in which Cre is expressed, allowing the conditional expression (reactivation) of the gene (Fig. 6C).

3.3.2. Cre-Expressing Transgenic Lines

The extent to which conditional gene targeting can be applied in biomedical studies depends at a technical level on the availability of Cre transgenic lines expressing Cre at sufficiently high levels under strict cell-type-specific and/or inducible control. At present, a wide number of Cre transgenic animals is available worldwide (see Note 24).

3.3.2.1. Generating Cell-Type-Specific Cre-Expressing Transgenic Mice

Both conventional transgenesis (see Subheading 3.1.) and targeted transgenesis (15) have been used for generating Cre-expressing transgenic lines. Cell-specific expression of Cre can be achieved by certain promoter/ enhancer combinations. The choice of promoter/enhancer combination should also be based on developmental timing of expression, because even transient Cre-expression during development or in progenitor-cell types would result in generalized excision in the adult. In our lab, we have generated and characterized a collection of various Cre-expressing mice, which allows gene targeting in T cells, myeloid cells, hepatocytes, follicular dendritic cells and B cells, synovial cells, oligodendroglia, or oligodendrocytes (see Note 24).

3.3.2.2. Inducible Genetic Modifications

Inducible Cre expression is extremely useful in the analysis of the role of certain genes in adult animals, because it offers the opportunity of gene modification after the completion of the normal development of the organism or of its adaptive cell systems, such as the neuronal and the immune system. Although constitutive cell-specific Cre expression can be achieved with the appropriate combination of promoters and enhancers, temporal control of Cremediated recombination can be exercised by regulating either the transcrip-tional or the posttranslational level of Cre expression. The most widely used binary transcription transactivation systems are the tetracycline-dependent regulatory systems. The tetracycline-inducible gene-expression system uses the DNA-binding domain of the tetracycline repressor (TetR) protein and the VP16 transcriptional activator domain, which are fused together. There are two versions of this system (45). In the original system, the transactivator (tTA) cannot bind DNA when tetracycline is present ("tet-off"), whereas in the modified version, the "reverse tTA" (rtTA) binds DNA only when the tetracycline is present ("tet-on"). A precise control on Cre expression can be accomplished by tissue-specific expression of the transactivator and administration of the inducer. Both tetracycline and ecdysone have been successfully used in transgenic mice (46,47).

Posttranslational control can be achieved by fusing Cre with a mutated ligand-binding domain (LBD) of steroid hormones that only bind synthetically produced steroid-hormone analogues. The fusion protein can be expressed under the control of a constitutive cell-specific promoter, but it remains in the cytoplasm. Administration of the synthetic analogue, such as Tamoxifen or RU-486, causes the translocation of the fusion protein to the nucleus where Cre gains access to loxP-flanked DNA targets (48-50).

3.3.2.3. Functional Characterization of Cre-Expressing Transgenic Lines

The efficiency and specificity of Cre-mediated recombination need to be tested at the cellular level using a transgenic reporter mouse line. Cre-express-ing mice should be crossed to a reporter line carrying a reporter gene such as P-galactosidase (P-gal), green fluorescent protein (GFP), or placental alkaline phosphatase (PLAP), in an inactive form, which, however, can be reactivated by Cre-mediated recombination. This recombination is usually achieved by the insertion of loxP-flanked inhibitory sequences between a ubiquitously expressed promoter and the reporter gene. By crossing the reporter line with a Cre line, the inhibitory sequences are removed only in Cre-expressing cells, thus allowing their identification. We have routinely used a Z/EG double reporter (lacZ/EGFP) mouse line (51) and a knockin lacZ into the ROSA26 locus as reporter lines (52). The activity of lacZ and GFP can be assessed both in tissue sections (12,13) or by flow cytometry (12,53) (see Note 25). Analysis at the DNA level (detection of recombined alleles by PCR or Southern blot analysis) is very informative about the percentage of cells that have undergone recombination (efficiency), but for the identification of such cells (specificity), especially in solid tissues, direct visualization is preferred.

4. Notes

1. Most transgenic mice are produced for two general purposes: (1) to study the control of gene expression, and (2) to study the effects of transgene expression in the intact animal. In the former experiments, transgenic mice should contain the entire gene of interest, as well as several kilobases each of 5'- and 3'-flanking DNA, i.e., using P1- or BAC-cloned large genomic fragments. In the latter experiments, tissue-specific promoters as well as regulatory sequences should be used to drive the expression of a transgene in a specific pattern in the mouse. However, the transcriptional efficiency of transgenes is almost always influenced by the activation status of the neighboring chromatin at the insertion site. At present, position-independent expression of transgenes can only be obtained using specific ds-acting DNA elements called locus control regions (LCRs) (54,55).

2. When designing a tissue-specific transgene, the TBASE database for transgenic mice can be searched at http://tbase.jax.org.

3. The classic preparation of plasmid DNA is a time-consuming approach and includes the use of CsCl gradient centrifugation. Lately, several commercial kits have appeared on the market allowing easy and fast isolation of plasmid DNA using disposable chromatography columns. We recommend starting with a commercial kit and using the CsCl gradient method as an alternative.

4. The concentration is adjusted on an agarose gel by comparison to an older fragment of similar size that has been used successfully in previous experiments.

5. Avoid touching the blunt end of the injection pipet with fingers when preparing them, since it could contaminate the DNA solution and lead to egg damage.

6. If the pronuclei are small, it may help to bring the embryos back to the incubator for an hour and start the injection process later. If the embryos are left for too long at 37°C the two pronuclei will have fused and injection will be impossible.

7. It is also possible to culture the injected eggs overnight in vitro to the two-cell stage and then transfer them following the same procedure. However, in vitro culture may decrease embryo viability.

8. Rupture of the bursa often causes bleeding, which can obscure vision. To avoid this, apply a drop of a 0.1% solution of adrenaline directly on the bursa just before tearing it. This approach has no apparent adverse effects and prevents all bleeding from the ruptured bursa.

9. The narrow part of the transfer pipet should be 2-3 cm in length and 120-180 ^m in external diameter, i.e., just larger than one egg and smaller than two. The presence of few bubbles after the eggs in the transfer pipet allows monitoring of the transfer procedure and ensures that all of the embryos have been successfully injected.

10. The quality of the DNA preparation at this stage is appropriate for slot-blot hybridization analysis, which provides a quantitative estimation for the copy number of the transgene.

11. The dissolved DNA can be directly used for PCR analysis. However, if the DNA is to be used for Southern hybridization analysis, a few more steps are included: after step 2, the samples should be digested with 1 ^L of 10 mg/mL RNase A for

1 h at 37°C. Steps 3 and 4 are repeated once more and are followed by an extra step of purification using 0.5 mL of chloroform/isoamyl alcohol.

12. We use the "pL2-neo" plasmid as a source of PMC-neo-poly(A) neomycin resistance cassette (kindly provided by Dr. Vasso Episkopou) as well as the "pNT" plasmid (56) bearing a herpes simplex virus thymidine kinase expression cassette.

13. We tend not to split PMEFs in high-dilution ratio, because in lower dilution they grow better for a longer period of time. Although it is hard to say how many passages they can sustain before they become senescent, their life span in culture is definitely smaller compared to stable lines used for feeders such as STO cells.

14. Inactivated feeders can be kept in frozen stocks as their active form. These stocks can prove very handy in case of emergency.

15. There are various methods for the DNA preparation used for gene targeting (e.g., kits using columns, and so forth). In our lab the CsCl preparation has worked the best since it delivers high-quality DNA without breaks, something very critical for the success of the targeting.

16. Use the minimum concentration of G418 that can kill all untransfected cells within 5-6 d. This concentration should be determined in advance by titration for each specific ESC line; usually it corresponds to 150-250 ^g/mL active concentration.

17. Another cryoprotectant medium for freezing, which is considered the economic way, consists of 25% FCS and 10% DMSO in ESC culture medium.

18. We find SealPlate the most efficient treatment. However, there are alternatives, such as Parafilm, small caps per well, or addition of 100 ^L of sterile, light paraffin oil to each well.

19. Alternatively, another method can be used to produce larger numbers of available synchronized blastocysts. In this protocol, superovulated immature females are used in the place of adult females. The females are again C57BL/6, 4-6 wk old, and they are mated with adult C57BL/6 males following the superovulation protocol in Subheading 3.1.3.

20. The time needed for the cell trypsinization, and for adherance in the preplating just before injection, varies among lines of ESC used. If not familiar, monitor cell detachment/attachment to the substrate following incubation for 3 min or 30 min, respectively.

21. The number of cells injected per blastocyst depends partly on how advanced developmentally the blastocyst is (early to fully expanded); it also depends on the condition of the cells to be injected. If more cells are expected to die, then the number increases.

22. Although we do the transfer to the uterus unilaterally, it is not prohibitive to transfer embryos into both uteri horns. Some laboratories practice this type of transfer, but to our experience the unilateral mode saves time without affecting the pregnancy outcome.

23. We use the "pEasy Flox" vector containing positive and negative selectable markers as well as 3-loxP sites (kindly provided by the group of Dr. Klaus Rajewsky) for the construction of conditional gene inactivation vectors.

24. Lists of currently available Cre-lines can be found at the following World Wide Web sites:

http://www.rnshri.on.ca/nagy/Cre.htm

This web site is maintained by Nagy's lab, and it contains an excellent list of almost all Cre lines that have been made.

http://www.fleming.gr/molgen/animal_resources.htm

This is our web site containing a detailed description of all the Cre-transgenic lines as well as TNF/TNFR transgenic lines and targeted mutant mice that have been generated and characterized by our lab.

• gttp://jaxmice.jax.org/jaxmicedb/htmL/transgene.shtml

This is the official web site of The Jackson Laboratory, listing all the Cre lines that can be purchased.

25. Although there is no direct evidence from our laboratory for intraline variability of Cre-expression and recombination efficiency, this is a parameter that should always be examined in each Cre-line. Furthermore, the recombination efficiency of a certain Cre-line should not be considered invariable for different loxP-flanked target-genes and should be assessed for each target gene separately.

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