1. Linear DNA has been shown to integrate into the germline more efficiently than circular forms (52). Prokaryotic vector sequences have been shown to reduce drastically the expression of eukaryotic transgenes (53). It is therefore necessary to remove as much vector sequence as possible from the transgene prior to microinjection. We have not experienced problems with expression from transgenes containing vector polylinker sequences, but these should be removed as far as possible.
2. Many modern pUC-derived vectors, such as the pBluescript (Stratagene, Cambridge, Cambs, UK) or pGEM (Promega, Southampton, Hants, UK) series, contain the lacZa region, allowing the use of a-complementation for blue/white screening during cloning procedures.
3. Problems of recombination may be experienced particularly when working with DNA fragments that contain repetitive sequences or have an irregular structure.
Characteristically, cloning steps will result in a very high proportion of recombinant clones that are truncated or rearranged. Initially, it may be worthwhile simply to alter the bacterial host. Strains that carry the recA mutation, for example, DH1, DH5a, or XL1-Blue, are deficient for homologous recombination. Certain strains, such as SURE® cells (Stratagene), carry additional mutations (e.g., recB, recJ, and sbcC) that interrupt specific pathways for the repair of DNA that is capable of forming irregular structures, such as inverted repeats and Z-DNA, or undergoes plasmid-by-plasmid recombination. An alternative approach that we have found to be very successful is to employ a lower copy number vector. Plas-mids may vary considerably in their copy number, and this is partly dependent on the origin of replication that they contain. Potentially, higher copy number vectors may be more prone to interplasmid recombination, and lowering the number of copies per cell may effectively stabilize an otherwise unstable plasmid. The majority of modern pUC-derived cloning vectors (e.g., pBluescript or pGEM) carry derivatives of the pMB1/ColE1 origin of replication and are maintained at copy numbers between 500 and 700/cell. pBR322 and its derivatives, for example, pPolyIII (13), also carry the pMB1 replicon, but have a much lower copy number (15-20/cell). Plasmids that are based on the pSC101 origin have only 1-5 copies/cell and may be the most suitable, although DNA yields are necessarily much lower and these plasmids cannot be amplified using inhibitors of protein synthesis (9). Alternatively, BAC or PAC vectors are thought to be extremely reliable for the cloning and propagation of unstable inserts (11,12).
4. The IRES sequence, derived from the picornaviridae family of viruses, which includes encephalomyocarditis virus (ECMV), foot and mouth disease virus (FMDV), and poliovirus, is able to initiate direct ribosome binding and protein translation (54). This is an alternative mechanism to the classical model of translation, which involves ribosome entry at the 5'-cap structure of a mRNA molecule (55). Potentially, an IRES-linked reporter gene can be introduced at any location within the transcribed region of the gene of interest, leading to the translation of both open reading frames from one mRNA (56).
5. There are two elements important for polyadenylation: a hexanucleotide poly(A) signal (AAUAAA) located 10-30 nucleotides 5' of the poly(A) site and 3' U-rich sequences. If these sequences are not present in the reporter construct or have been removed during deletional analysis, an artificial one will have to be added. Most commonly used are the transcriptional termination and polyadenylation sequences from the SV-40 early region, SV-40p(A), (see ref. 5). It must be noted that heterologous poly(A) sequences are not always advantageous. In the case of Hoxb-4, stronger transgene expression is seen in the presence of its own tran-scriptional termination sequences than with those of SV-40 (Morrison and Krumlauf, personal communication).
6. Certain genes have high rates of mRNA turnover, for example those encoding gene products required rapidly, but for a short time, which may lead to very weak levels of reporter gene activity. The rapid turnover of mRNA is generally conferred by AU-rich elements (AREs) consisting of repeated AUUUA sequences and located in the 3'-UTR of the gene (57,58 and references therein). We have found that it is possible to increase the activity levels from constructs containing AREs by the addition of the SV-40p(A) sequence to the 3'-end of the lacZ gene (Summerbell and P. W. J. R., personal communication).
7. To simplify analyses using constructs of this type, the promoter used should be either inactive or unable to direct any specific expression in the absence of regulatory regions.
8. In this case the staining protocol will need to be optimized for the two reporters. Since AP is a membrane-associated protein, staining for AP activity will tend to mask any intracellular staining for P-galactosidase. The most suitable lacZ reporter in this case is probably the nuclear localized form. Another option is to use immu-nohistochemical detection methods to distinguish better between the activities of AP and of P-galactosidase, either in whole mount or sectioned samples.
9. We generally use Neuro 2a cells (a mouse neuroblastoma cell line), which we have found to have a high efficiency of transfection and to have worked with a range of reporters.
10. Suitable negative and positive control plasmids can be included, e.g., the basic construct without the reporter gene and pSV-Pgal (Promega), respectively.
11. Depending on the efficiency of transfection, between 1 and 15% of the cells should exhibit a high level of reporter gene staining, usually visible within 1 h. Longer periods may be required for weakly expressing constructs.
12. Use clean, dust-free labwear throughout, preferably rinsed with filtered water. Filter solutions through a 0.22-^m filter to prevent the addition of particulate matter to the DNA sample. Wear powder-free gloves or rinse powder-coated gloves with water prior to handling samples.
13. It is important to start with a large amount so you can afford to lose some on the way. The more concentrated the final DNA preparation is, the more any impurities will be diluted at the microinjection stage.
14. By using a slightly higher percentage of LMP agarose, the migrating band can be tightened, reducing the sample volume for subsequent Agarase digestion. Make sure the fragment has migrated well into the LMP Agarose to avoid the carryover of normal agarose, which will not be digested by P-Agarase. Ensure sample is fully molten. Allow sufficient time for temperature equilibration.
15. This is the minimum time required for complete Agarase digestion by this method, although samples may be incubated for extended periods (e.g., overnight) without degradation of the DNA.
16. An optional step may be included to remove any undigested carbohydrates. Adjust the salt concentration of the sample for alcohol precipitation by the addition of sodium acetate to a final concentration of 0.3 M. Chill on ice for 10-15 min and centrifuge at 15,000g for 15 min to pellet any undigested carbohydrates. This is unnecessary if Agarase digestion is complete.
17. Perform the extractions in a suitable volume, taking care to leave all of the interface and some aqueous phase behind each time. Centrifugation at 15,000g for 5 min is sufficient to ensure adequate phase separation. Diethyl ether has a lower density than that of water and partitions as the upper phase. Residual ether can be evaporated by leaving the uncapped sample in a fume hood for 5-10 min.
18. Phenol is corrosive and can cause severe burns. Chloroform is an irritant and a carcinogen. Work with these chemicals should be performed in a fume hood with appropriate protection. Irrigate areas of the skin that come into contact with phenol with a large volume of water or PEG-300. Wash with soap and water. Seek medical advice if necessary.
19. We routinely run serial dilutions of our samples against known amounts of digested X DNA (HindIII, BstEII, or EcoRUHindlII) run in several control lanes. For example, 485 ng of X-HindIII cut DNA will yield markers ranging from 23 to 1.25 ng. It is advisable to compare the fluorescence of the transgene fragment with other DNA fragments of similar size, since the relationship between these two parameters is not strictly linear. For this reason, we also run standards consisting of similar-sized microinjection fragments that have yielded good frequencies of transgenics in previous experiments.
20. Excessively high concentrations of DNA are detrimental to the embryo and reduce survival rates. We routinely use DNA at a concentration of 1.5-2 pg/mL.
21. Glass liquid-scintillation vials are good containers for bulk processing of embryos. When you wish to keep individual embryos separate (e.g., for subsequent transgenic diagnosis), plastic multiwell plates (24-well, Sigma-Aldrich) can be used.
22. Plastic, disposable Pasteur pipets (e.g., Pastettes, Alpha Laboratories Ltd., Eastleigh, Hants, UK) are useful for the handling of embryos and can be cut to the required diameter.
23. Use a large enough volume of fixative to avoid significant dilution by carried over PBS. Alternatively replace the fixative once the embryos have been transferred.
24. The type of fixative used depends on personal choice and subsequent histological procedures. Many researchers use a solution containing 1% (w/v) formaldehyde, 0.2% (w/v) glutaraldehyde (5), or 4% (w/v) paraformaldehyde. We prefer to use Mirsky's Fixative (National Diagnostics), since it is simple to make up (from a 10X concentrate and 10X buffer), has a long shelf life (1 mo at 4°C), and is relatively nontoxic. It is a mild fixative, leaving the embryos soft and easy to section.
25. By performing the P-galactosidase assay at the optimal pH for the bacterial enzyme (pH 7.3), there should be relatively little activity of the endogenous mammalian lysosomal P-galactosidase (pH optimum between 3.0 and 6.0). However, during prolonged incubations, Tris-HCl (pH 7.3) should be added to the stain solution to stabilize the pH of the phosphate buffer. The effects of endogenous P-galactosidase activity can also be reduced by performing the staining reaction at lower temperatures.
26. For preliminary investigations where the expression patterns of transgenes are to be compared, staining conditions (i.e., time and temperature) should be kept as constant as possible. Prolonged staining, especially at 37°C, will lead to a gradual degeneration of sample tissue and may cause a loss in morphological resolution with sectioned samples. In this case, it is worthwhile to keep the staining conditions as conservative as possible.
27. For AP and P-galactosidase/AP double staining, conditions should ideally be optimized for the particular construct and developmental stage. Staining/heat-inactiva-tion times and the addition of AP inhibitors, e.g., 1 mM levamisole (l[-]-2,3,5,6,-tetrahydro-6-phenylimidazo[2,1-b]thiazole, Sigma) may be tested to reduce the level of endogenous alkaline phosphatase activity present in the embryo (59).
28. Use tightly capped storage tubes to prevent ethanol loss by evaporation. During long-term storage, change the solution periodically.
29. Care is required throughout this procedure to avoid sample contamination with plasmid DNA or PCR products. Preferably perform the method in a separate, clean environment. Keep all solutions separate from plasmid manipulations and store in aliquots. Use separate pipets for manipulations and/or use filtertips (e.g., FilterPro, Rainin Instrument Co., Luton, Beds, UK) to reduce the risk of contaminating pipet barrels.
30. We have found that the two boiling steps prior to and after digestion significantly increase both the sensitivity and quality of the PCR reaction. This is most conveniently achieved by placing the samples in a water bath at 60-70°C, which is then turned up to boiling for 5 min. The water bath may then be turned down to 55°C, and the samples allowed to cool prior to the addition of proteinase K.
31. It is useful to make up a large volume of 1X diagnostic PCR mix containing buffer, MgCl2, dNTPs, primers, and water. This can be stored at -20°C in aliquots of 20100 reaction volumes to which only Taq polymerase and template need to be added.
32. For lacZ/SV-40 transgenes, we routinely use the primers LZ3 (5'-GCGACTTCCAGTTCAACATC) complementary to the minus strand of lacZ and STB (5'-GATGAGTTTGGACAAACCAC) complementary to the plus strand of the SV-40 poly(A). As a qualitative internal control, we use MGP1 (5'-CCAAGTTGGTGTCAAAAGCC) and MGP2 (5'-CTCTCTGCTTTAAGGAGT CAG), specific to the endogenous myogenin gene (6). These primer pairs amplify fragments of 590 and 172 bp, respectively. Conditions used for the reaction are 94°C/3 min, 94°C/30 s, 55°C/30 s, 72°C/30 s for 26 cycles at a final Mg2+ concentration of 1.5 mM.
33. What is known about the transcriptional regulation of the gene of interest? Have in vitro studies identified regulatory elements that should be included or eliminated from the transgene or might be involved in particular aspects of expression? Can sequence comparisons pinpoint any areas of conservation within noncoding regions that may have regulatory functions?
34. The TRANSFAC transcription factor database (60) is a useful reference source of transcription factors and their associated DNA target sequences. The database is available with an online search facility via the internet (http://transfac.gbf-braunschweig.de/).
35. The expression patterns obtained from any given construct can be extremely variable, particularly when working with small fragments of DNA out of their normal genomic context. For this reason, it is important to compare a number of transgenic animals (ideally six or more) for a particular construct, so that variabilities and consistencies in the expression pattern can be assimilated.
36. For example, denervation of muscles in adult mice carrying a lacZreporter driven by regulatory sequences from the acetylycholine receptor a-subunit leads to the transcriptional activation of the transgene, but to a decrease in p-galactosidase staining probabaly because of an increase in proteolytic activity (61). This discrepency is not seen when the gene encoding chloramphenicol acetyltransferase is used in an essentially identical transgene construct.
37. LacZmRNA and protein may persist for 3-4 d in transgenic animals. Therefore, a reporter gene may continue to show activity in cell lineages long after the reporter and endogenous gene have been switched off in them (62). This can be particularly problematic at early stages of development when rapid growth is most evident.
38. We have had the greatest experience with the p-globin and hsp68 promoter systems. The advantages of P-globin over hsp68 are that the minimal promoter is small, simple, and well defined. Containing both TATA box and initiator elements, it is able to work with a wide range of enhancers to generate faithful patterns of reporter gene expression. One disadvantage of the P-globin promoter is that compared to hsp68, it is relatively feeble, and with weak enhancers, it may give a low percentage of expressing transgenic offspring.
39. Several researchers, including ourselves, have experienced a pattern of consistent, but ectopic expression in the ventral neural tube of transgenic embryos with hsp68 promoter constructs. This has been attributed to an element in the hsp68 promoter that is able to interact with enhancer elements juxtaposed to it (63-65), and at some frequency, we have seen this pattern of expression with many different enhancer constructs. It is particularly useful as a control for effective transgenesis in situations in which essential transcriptional regulatory elements in the enhancer under the test have been inactivated by mutation.
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