lacZ Transgenic mice to monitor gene expression in embryo and adult

Brain Research Protocols 3 Ž1998. 54–60

Protocol

lacZ Transgenic mice to monitor gene expression in embryo and adult Andrea Schmidt a

a,1

, Kirsten Tief a,1,2 , Alessandro Foletti a , Agnes ` Hunziker a , Doris Penna b Edith Hummler , Friedrich Beermann a, )

a,3

,

ISREC (Swiss Institute for Experimental Cancer Research), Chemin des BoÕeresses 155, CH-1066 Epalinges, Switzerland b Institute of Pharmacology and Toxicology, UniÕersity of Lausanne, Bugnon 27, CH-1005 Lausanne, Switzerland Accepted 23 April 1998

Abstract In transgenic experiments, lacZ can be used as a reporter gene for activity of a given promoter. Its main advantage is the ease of visualization in situ, on sections or in whole mount preparations, and the availability of simple protocols. In the following, we describe our procedure for detecting promoter activity in transgenic mice, including choice of lacZ vectors, generation of the transgenic mice, and analysis of expression. We had recently used this protocol to detect tyrosinase gene promoter activity in embryonic and adult brain. q 1998 Elsevier Science B.V. All rights reserved. Themes: Cellular and molecular biology Topics: Staining, tracing and imaging techniques Keywords: Transgenic mouse; lacZ; Promoter

1. Type of research Ø Analysis of promoter function Ø Identification of cis-acting elements Ø Detection of unknown areas of gene expression in whole embryos or whole organs Ø Whole mount staining of embryos Ø Cell lineage studies

2. Time required Ø Transgenic constructs: 1 week to 1 month Ø Production of transgenic mice Žfrom injection to adult founder.: 12 weeks Žto adult F1: another 8 weeks.

) Corresponding author. Fax.: q 41-21-652-6933; E-mail: [email protected] 1 Both authors contributed equally to the manuscript. 2 Present address: Perkin ElmerrApplied Biosystems, Cheshire WA3 7PB, UK. 3 Present address: Institute of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland.

Ø Analysis of mice by PCR: 1–3 days Ø Staining for b-galactosidase activity: 1–2 days

3. Materials Ø Mice: NMRIrHan mice, CB6F1 mice and B6D2F1 mice are commercially available Že.g., Harlan, Netherlands.. No special requirements for mouse maintenance and husbandry are needed. Ø Constructs: placZ Žobtained from G. Schutz, ¨ DKFZ, Heidelberg., placF and pnlacF Žobtained from J. Peschon, Immunex, Seattle, WA.. Ø PCR-primers: ordered from MWG-Biotech ŽGermany. or Microsynth ŽSwitzerland.. Chemicals were obtained from standard laboratory suppliers ŽMerck, Fluka or Sigma. unless otherwise stated. Ø X-gal ŽAppligene cat. No. 130 262.. Ø Bluo-gal ŽSigma cat. No. B-2904..

4. Detailed procedure An overview of the procedure is given in Fig. 1.

1385-299Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 2 1 - X

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Fig. 1. Scheme of the protocol. Numbering corresponds to the individual steps as described in detailed procedure and quick procedure. Ž1. A transgenic construct including promoter, lacZ gene and polyadenylation sequences is deliberated from vector sequences before injection. Ž2. Injection of DNA into fertilized oocytes and transfer to recipient females. Ž3. Identification of transgenic founders by PCR. Ž4.rŽ5. Brain or embryo are either fixed and stained directly, and sectioned thereafter. Alternatively, they are first sectioned, then fixed and stained. Ž6. It is feasible to clear stained embryos.

4.1. Constructs We have used two different basic constructs, placF and placZ. Construct placZ contains SV40 small T splice and polyadenylation sequences, placF mouse protamine sequences, including splice and polyadenylation sites. The presence of these introns permits detection of transgene expression using RT-PCR Žreverse transcriptase PCR. and might increase transgene expression w6x. Regulatory sequences are inserted 5X to the lacZ coding sequences into unique restriction sites. The 3X end of the upstream sequence should reside between the translational start ŽATG. and the transcriptional start site. By absence of a suitable restriction site, this can be created by PCR. In both placF and placZ, unique restriction sites should be maintained, which allow to deliberate the construct from vector sequences before injection into mouse oocytes. Construct pnlacF differs from placF by presence of a nuclear localization signal, thus allowing staining to be restricted to the nucleus Žsee Ref. w5x for discussion of nuclear vs. cytoplasmic X-gal staining.. In placF and pnlacF, the presence of a NcoI site Ž NcoI: CCATGG. encompassing the ATG allows an in-frame fusion within the coding sequences of the gene of interest. This is of major advantage when transcriptional start sites are not known or when intronic sequences are included. A failure of such fusions can be caused by presence of leader sequences causing e.g., secretion of the product, or hampered enzymatic activity of the fusion. It should be men-

tioned that presence of IRES Žinternal ribosomal entry site. sequences might circumvent this problem w32x. Independent translational start from the bi-cistronic message is achieved on the ATG of the lacZ gene. 4.2. Transgenic mice Production of transgenic mice followed standard procedures and will only be mentioned briefly w11x. Constructs were deliberated from vector sequences. Fragments were purified from agarose gels using glass powder and filtered through 0.2 m m filters. DNA was diluted to concentration of 3–5 m grml Žin 10-mM Tris, pH 7.5, 0.1 mM EDTA. by comparison to known standard and other transgenic constructs. Per construct, 100–300 fertilized oocytes were microinjected and transferred to pseudopregnant females, giving rise to 10–50 offspring, representing at least three transgenic founders. 4.3. Identification of transgenic mice lacZ transgenic mice were identified on DNA isolated from tail biopsies w2x. Mice were separated and marked, and 1–10 mm of mouse tail was cut and digested overnight at 568C in a tube containing 750 m l of 100 mM NaCl, 50 mM Tris–HCl ŽpH 8.0., 100 mM EDTA ŽpH 8.0., 1% SDS and 0.5 m grm l proteinase K. After brief shaking or vortexing, 250 m l of saturated NaCl Žabout 6 M. was

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A. Schmidt et al.r Brain Research Protocols 3 (1998) 54–60

added. Tubes were shaken by hand or vortexed thoroughly, and centrifuged for 10 min to pellet debris and protein. A total of 750 m l were transferred to a tube containing 500 m l of isopropanol. DNA was precipitated by shaking and mixing the two phases and pelleted by 2 min of centrifugation. After washing in 500–800 m l ethanol, tubes were allowed to dry for 10 min. DNA was dissolved for at least 2 h at 378C in 120–150 m l of 10-mM Tris ŽpH 8.0. and 1 mM EDTA ŽpH 8.0.. PCR amplification Ž1 cycle: 4 min, 948C; 30 to 40 cycles: 0.5 min, 948C, 1 min, 558C, 1 min, 728C; one final elongation of 10 min at 728C. was performed on 1–2 m l of DNA in 50 m l of 50 mM KCl, 10-mM Tris ŽpH 8.3., 2-mM MgCl 2 , 0.1-mM dNTPs, 0.01 nmol of each primer and 1.25 U of Taq polymerase ŽLife Technologies, Basel, Switzerland.. Following primers were used: lacZ-specific amplification: 5X -AACTTAATCGCCTTGCAGCA-3X Žq67–q 87; Ref. w14x. and 5X-GTGCATCTGCCAGTTTGAGG-3X Žq379–q 360; Ref. w14x.. The second primer Ždownstream. can also be used in combination with a primer specific for the promoter sequence. Amplification specific for SV40 splice and polyadenlyation sites: 5XGGCATTTCTTCTGAGCAA-3X Žq4506–q 4489 of SV40 DNA. 5X-CTTACTTCTGTGGTGTGA-3X Žq4679– q 4696 of SV40 DNA.. Amplification specific for placF and pnlacF using lacZ and mouse protamine sequence: 5X-TGGCTGAATATCGACGGTTTC-3X Žq2947–q 2967 of lacZ, Ref. w14x. and 5X-TGTTTTTCATCGGACGGTGGC-3X Žq1006–q 986 of mouse protamine 1, Ref. w12x.. Southern blot analysis and hybridization using randomly primed 32 P-labelled probes followed standard procedures. They are only needed to determine copy numbers, to corroborate the PCR result, or to identify possible presence of two or more independent transgenic integrations sites. As 32 P-labelled probes, we used either lacZ coding region or SV40 splice and polyadenylation sequences. 4.4. Whole mount staining for b-galactosidase actiÕity on embryos or total brain The procedure for staining consists of a fixation step Ž48C., a washing step Ždetergent at RT. and the staining itself Ž378C, waterbath.. Fixation and wash take about 2–4 h, the time for staining depends on the strength of the promoter, and is generally achieved after incubation over night. We used staged embryos or total brain, which were removed from the euthanized mouse. Embryos Žup to E13.5. were fixed for 10–30 min, larger embryos Že.g., E16.5. and adult brain was fixed for 45 min, then cut sagittally with a razor blade, and further fixed for another 45 min. Fixative was replaced by detergent wash ŽRT., which was changed once Žtotal 1.5 h.. Staining was performed at 378C in a water bath, with coloration appearing after 1–24 h. Staining solution was recovered and kept at

48C, stained embryos or organs were washed for 1–2 h in PBS and stored in fixative. Fixation: 4% paraformaldehyde ŽPFA. in PBS. We prepared a larger batch by dissolving the PFA in prewarmed PBS, controlled the pH Žwe used pH 7.2. and froze down aliquots of 10 ml. Detergent wash, final concentration: 0.1-M sodium phosphate buffer ŽpH 7.3., 2-mM MgCl 2 , 0.01% of sodium desoxycholate, 0.02% of NP40. We prepared a 2 = stock, by mixing 80 ml of 0.5 M sodium phosphate buffer ŽpH 7.3., 800 m l of 1 M MgCl 2 , 4 ml of 1% sodium desoxycholate, 80 m l of 100% NP40 and filling up to 200 ml with distilled water. Staining, final concentration: 1 = detergent wash Žsee above., 3.33-mM potassium ferricyanide, 3.33-mM potassium ferrocyanide, 0.66 mgrml X-gal and 20-mM Tris ŽpH 7.0.. In practical terms, we added per 15 ml: 7.5 ml of 2 = detergent wash, 500 m l of 0.1 M potassium ferricyanide, 500 m l of 0.1 M potassium ferrocyanide, 300 m l of 1 M Tris ŽpH 7.0. and 500 m l of 2% X-gal Ždissolved in dimethyl formamide, kept in polypropylene tubes at y208C.. The potassium salts were kept at 48C in the dark, and although it is often recommended not to keep them too long, they still worked fine after more than 8 months. Staining solution was made directly before use. Nevertheless, older staining solution can be filtered, stored at 48C in the dark and reused. When it was necessary to section already stained materials, organs or embryos were dehydrated through ethanol and embedded in methacrylate. The results using thin sections are not always satisfactory, especially when expression of transgene is weak. One alternative are vibratome sections which allow thicker sections Žsee for example Ref. w18x. We have successfully replaced X-gal as chromogenic substrate by Bluo-gal w29x. The reaction product precipitates in the form of small crystals and can be visualized by microscopic examination under polarized light. This is very sensitive and is compatible with counterstaining. The method has been described elsewhere in detail w1x. It is equally feasible to process X-gal stained embryos or organs for cryostat sections, after removal of fixative and ethanol by different washing steps. This was only performed for strong staining, and cellular resolution is weaker than with methacrylate or paraffin sections. 4.5. Staining of cryostat sections 4.5.1. Sections Embryos or organs Žbrain. were washed on ice in PBS, embedded in TissueTek and immediately frozen in isopentane cooled in liquid nitrogen. Either continue directly doing sections or store the frozen tissue at y708C. Sectioning was performed at y188 to y208C using silanecoated slides. Two to three sections were collected per slide.

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4.5.2. Fixation and staining Fixation solution contained 0.1 M of sodium phosphate ŽpH 7.3., 0.2% glutaraldehyde Žkept in aliquots as 25% stock at y208C.. Add a few drops of freshly prepared fixation solution on each 10-m m section. After 1–3 min, stop reaction by brief washing in PBS. Remove excess liquid with wipe and place slides in a box containing humidified paper towel. Add 50–80 m l of freshly prepared staining solution Židentical to whole mount staining. onto each slide and incubate overnight in the dark at RT. The next day, slides were washed briefly in water. If necessary, sections were counterstained Ž3 min in 0.25% eosin in water. processed through ascending ethanol and Xylol Žor Toluol. and mounted in Eukitt. 4.6. Clearing of X-gal stained embryos Different protocols for clearing stained embryos are available. We have used benzyl benzoate for embryos Žbut not yet for organs., which rendered them transparent. However, they cannot be sectioned later on. Embryos were processed through ethanol Ž30%, 50%, 70%, 99% for 30 min each. and transferred into benzyl benzoate: benzyl

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alcohol Ž2:1.. They were kept in this solution, but we photographed them directly, since blue stain disappeared after prolonged storage.

5. Results The reporter gene lacZ has been repeatedly used in transgenic experiments. Detection of transgene expression by histochemistry and X-gal staining has been reported for a wide variety of applications. Besides the interest in defining the 5X regulatory regions of certain genes, others have used this approach to mark cell lineages, or to detect new hitherto unknown areas of expression. Papers on lacZ staining in transgenic animals Žand in neural development andror brain. are appearing continuously and only a few recent ones can be cited as examples w15,31,33,35x. We were interested in tyrosinase promoter activity during development w29x. We have cloned tyrosinase 5X sequences into both the placZ and the placF vectors w29x. Out of 150–200 injected and transferred oocytes, we finally obtained three transgenic mice for each construct. These transgenics were identified by Southern blot analysis Žnot

Fig. 2. Detection of b-galactosidase activity in tyrosinase-lacZ transgenic mice. ŽA. Whole mount staining of adult transgenic brain following sagittal section. o s olfactory bulb, c s cortex. ŽB. Staining of sagittal brain section with positive signal in substantia nigra Žsn. and in fibres ascending to epithalamus Žarrows.. ŽC. Section through eye of X-gal stained E12.5 embryo. ŽD. E12.5 embryo with strong signal in telencephalon Žte. following clearing. Signal in eye is not visible due to presence of pigment.

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shown. and F1 offspring was identified by PCR. In our hands, about 16% of transferred oocytes survive the manipulations and are born. Of these, again 16% are transgenic thus yielding an overall efficiency of roughly 2.5%. Transgenic males were mated to non-transgenic females to obtain staged embryos Žthe day of vaginal plug was counted as E0.5., which were stained with X-gal Žor Bluo-gal. w29x. When embryos were cleared and made transparent, 3-dimensional distribution of stain was even better detected ŽFig. 2D.. Other embryos stained with X-gal were sectioned ŽFig. 2C.. Bluo-gal staining was visualized under polarized light, and positive signal is easily detected as white birefringent crystals w1,29x. In transgenic embryos, b-galactosidase activity was detected along the entire neural tube, with most prominent expression in developing telencephalon. Staining was equally observed in the developing retina. These results suggested a possible expression of tyrosinase and a corresponding function in neural development. In brain of adult transgenic mice, we equally detected positive staining for b-galactosidase. Moreover, we demonstrated expression of tyrosinase mRNA and protein in adult brain of non-transgenic animals w28x. Staining was detected in several specific areas of fore- and midbrain, cortex, olfactory system, hippocampus, epithalamus and substantia nigra w30x. Here, we either used staining on whole adult brains ŽFig. 2A. or stained cryostat sections ŽFig. 2B.. In general, the reduced fixation period allows a more sensitive detection w30x. However, the overall intensity might decrease in these thinner sections.

6. Discussion 6.1. Troubleshooting There are several reasons for failure to detect any staining in organs or embryos, when this is expected from the endogenous expression pattern. ŽA. Background staining or no staining could be due to a technical problem Žfixation, sensitivity. which might be improved by changing parameters Žfixation time, fixative, staining on sections.. Normally, whole mount staining of younger embryos Žup to E12.5. does not result in background staining. Background is often due to incomplete fixation or old fixative, or the pH of the solutions. In addition, enough staining solution should be used to buffer any pH changes caused by the embryo. For adult organs, it is equally important to control pH and maintain fixation times as a compromise between decreasing sensitivity and increasing background. Nevertheless, we did not achieve convincing staining results in organs like kidney or gut, which apparently have a rather high level of endogenous b-galactosidase activity. When weak promoters are used, it is possible to use cryostat sections which are stained afterwards.

ŽB. No X-gal staining might be detected on whole mount or sections even if expression of the transgene can be detected by RT-PCR. According to a recent report, use of specific anti-b-galactosidase antibodies might be more efficient than X-gal staining w7x. We have tested this antibody Žpolyclonal rabbit anti-b-galactosidase antibody, Chemicon, Temecula, USA. on cryostat sections of brain from tyrosinase-lacZ transgenic mice. When comparing lacZ histochemical staining and immunofluorescence we could not detect any difference in sensitivity Žnot shown.. Nevertheless, immunostaining might be considered as an additional tool for detection of expression from weak promoters. ŽC. The construct does not contain sufficient regulatory sequences for expression in vivo. This requires further analysis of the regulatory region Žfor example DNAse I hypersensitive sites. and the inclusion of more 5X sequences Žsometimes also intronic or 3X sequences.. ŽD. The transgenic insertion site might reside in a silent chromatin region. Several independent transgenic lines should therefore be analysed Žor dominant control regions included.. In addition, independent transgenic lines are needed to exclude any false interpretation due to ectopic expression of the transgene. Staining should be confirmed in several independent transgenic mouse lines. ŽE. No expression or very weak expression might be seen when in-frame fusions are used Žsee Section 4.1.. The fusion might result in a protein which is too long, and the C-terminal lacZ is not completely translated or not anymore active. Presence of signal sequences Že.g., for secretion. in the N-terminal part of a protein might result in secretion of the fusion protein. We had constructed an in-frame tyrosinase-lacZ fusion gene, which resulted in very weak staining in only one line w29x. This construct still contained the presumed 19 amino acid signal sequence of mouse tyrosinase, which possibly led to secretion of the fusion protein. Such problems might be circumvented in future by the use of an internal ribosome entry site ŽIRES. which is placed in front of the lacZ w32x and enables independent translational initiation. Before starting transgenic analysis of a promoter-lacZ fusion construct, this promoter should have been tested in vitro, by transfection analysis. For the tyrosinase promoter, we had used fusions to the reporter gene CAT which demonstrated that the 5X region we used was at least sufficient for specific expression in cultured cells w16x. We also tested the tyrosinase-lacZ constructs in transient transfections into B16 cells Žunpublished data.. Few blue cells were detected, but this nevertheless assured that the construct was functional. For studies on gene regulation and to define the importance of specific cis-acting elements, generation and analysis of transgenic mice carrying a lacZ reporter gene is the most suitable method. To define the expression pattern of an endogenous gene, in situ hybridization is normally used Žbesides RT-PCR or Northern blot experiments.. Neverthe-

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less, information on spatial expression is less amenable and limited by the size of the embryo in whole mount in situ hybridization. Thus, once a transgenic line is generated, where lacZ expression reflects endogenous expression, spatial expression of the gene of interest can be followed easily. Moreover, such a transgenic line can be used to monitor induced changes in gene expression, as demonstrated for fos–lacZ transgenic mice w25x. In addition, it is feasible to stain cryostat sections for b-galactosidase activity, and to process thereafter to immunohistochemistry on the same section, which allows to characterize neuronal cell types more precisely. Some examples for such dual staining were published for neurofilament-M w34x, dopamine b-hydroxlase w20x or the neuronal marker Q502, which has been used following grafting of lacZ-expressing neural cell lines w26x. 6.2. AlternatiÕe and support protocols Protocols for staining for b-galactosidase activity can be found in the literature w4,11,17,21,36x or on the internet w19x and might be useful for comparison. Several more refined applications have been used over the last years. The following is not an exhaustive list, and the interested reader should refer to literature databases for further information. For example, lacZ transgenic mice have been used for studying mutations, based on the recovery of chromosomally integrated lacZ-containing plasmids Žor phages. w3,22x. In gene targeting experiments, endogenous sequence is commonly replaced by the neomycin resistance gene driven by an ubiquitous promoter. Expression of the targeted allele can generally not be analysed. However, the inclusion of a lacZ reporter gene, which is placed in frame with the endogenous sequence, allows to follow both the normal expression pattern of the targeted gene and to analyse the phenotype of the mutation. If the targeted gene is expressed in the embryonic stem cells, the lacZ gene and the neomycin resistance gene can be fused to generate a single open reading frame w13,36x. Several gene targeting experiments have been published taking advantage of the lacZ marker Žfor example Refs. w8,27x.. In the neurotrophin-3 Ž NT-3 . gene knockout, the lacZ marker convincingly demonstrated that the histochemical expression pattern was identical to the expression of the endogenous NT-3 gene, as shown by in situ hybridisation. Moreover, the same pattern was maintained in the homozygous mutant situation, thus excluding effects of NT-3 on its own expression w9,10x. In experiments devised to analyse NT-3 in trigeminal neurons, cryostat sections were stained first for bgalactosidase activity and then analysed by immunohistochemistry for expression of neurofilament-M w34x. lacZ is also used to trap new genes and expression domains following random insertion of specifically designed vectors into embryonic stem ŽES. cells. Activation of lacZ indicates integration into a gene or a functional

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promoter region. ES cells are injected into blastocysts and b-galactosidase expression is monitored in chimeric embryos. In gene trap experiments, this can also be used to create mice homozygous for the mutation. Since the vector sequences are known, the corresponding gene can be isolated and further characterized w23,24,36x.

7. Quick procedure 1. The promoter region of interest is cloned into one of the lacZ vectors. 2. Transgenic mice are generated by microinjecting the construct into fertilized oocytes. 3. Transgenic founder mice are identified by Southern blot analysis or PCR on DNA isolated from tail tips. 4. Staged embryos or adult brain Žor other organs. are fixed and stained with X-gal or Bluo-gal Žand, if desired, sectioned afterwards.. 5. As alternative to Ž4., embryos or brain are sectioned on a cryostat and stained afterwards. 6. It is possible to clear such stained embryos to render them translucent.

8. Essential literature references Refs. w1,4,9,10,17,18,24,29 x.

Acknowledgements We are grateful to Ian Jackson, Steve Morley and Adriano Aguzzi for providing their protocols. This work was supported by grants from the Swiss National Science Foundation and the Swiss Cancer League.

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