k./ 1992 Oxford University Press
Nucleic Acids Research, Vol. 20, No. 21 5655 -5660
The developmental regulation of the human -globin in transgenic mice employing j3-galactosidase as a
gene
reporter gene
M.D.Pondel, N.J.Proudfoot, C.Whitelaw and E.Whitelawl,* Sir William Dunn School of Pathology, Oxford University, Oxford OX1 3RE, UK and 1Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia Received August 5, 1992; Accepted September 25, 1992
ABSTRACT We have investigated the developmental and tissue specific expression of the human embryonic t-globin gene in transgenic mice. A construct containing 550 bp of t-globin 5' flanking region, fused to a ,Bgalactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like a positive regulatory element (aPRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire t-globin gene linked to the aPRE or the ,3LCR. Data showed that 12% of mice generated from eggs injected with P-promoter/lacZ/aPRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire t-globin gene linked to the aPRE or the ,BLCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, posssibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that t-globin promoter activity was most pronounced at 8.5 - 9.5 days of development and was restricted to erythroid cells. By 15 days of development, no t-globin promoter activity was detected. These results suggest that the aPRE can direct high level expression from the t-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human f-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the P-globin gene or 3' of the cap site do not appear to be necessary for correct P-globin developmental regulation. INTRODUCTION The hemoglobin molecule is a tetrameric protein containing two a like globin chains and two ,B like globin chains. The human cx-globin gene cluster consists of three fuctional genes arranged 5'-v2-a2-al -3' at the tip of chromosome 16p. The human ,*
To whom correspondence should be addressed
globin cluster consists of five functional genes arranged 5-e-GyA,y-6-3-3' on the tip of chromosome lp. The genes within each cluster are regulated in a tissue and developmental stage specific manner to produce embryonic (r2E2, a2E2 and t2-y2), fetal (a2,y2) and adult (a2a2 and a2j32) hemoglobin. In the human ,B cluster, the embryonic E-globin gene is expressed from about 3 to7 weeks of development followed by a switch to expression of the two adjacent fetal 'y-globin genes. An additional switch from -y-globin expression to f-globin gene expression occurs at birth (1). In the ca cluster, a single switch from t2 to c2 and a l expression takes place at 6-7 weeks of development (2). The mechanism(s) responsible for the coordinated regulation of these two physically separated gene loci is hypothesized to occur at the transcriptional level (3). Transgenic mice have provided a useful system for the study of human globin gene regulation. When a 4 kb fragment containing the entire 3-globin gene is introduced into the genome of mice, correct developmental and tissue specific expression is attained. Expression, however, is less than 5% of endogenous globin expression and dependent upon the site of integration in the genome (4-6). Transgenic mice containing a fragment that includes the entire human a-globin gene show no expression of the introduced gene (7-9). When the ax- and ,3-globin genes on intact chromosomes 16 and 11 are transferred to human X mouse erythroleukemia cells (MELCs), their level of expression is similar to that of the endogenous mouse globin genes (10-13). These results suggest that additional cis acting regulatory sequences remote from the a- and 0-globin gene cluster are necessary to achieve high levels of position independent a and j-globin expression in transgenic mice. Studies have shown that high levels of human ,B-globin gene expression in stably transfected cell lines and transgenic mice is dependent on DNA sequences located 5-20 kb upstream of the human j3-globin locus. These sequences, collectively referred to as the locus control region (LCR) can direct high level expression of both at- and ,B-globin genes in a copy dependent, position independent manner in transgenic mice (7,9,14,15). More recently, an LCR like a-globin positive regulatory element (aPRE) located 30-40 kb upstream the human t-globin gene
5656 Nucleic Acids Research, Vol. 20, No. 21 has been identified. Like the ,BLCR, the aPRE is capable of directing high levels of a-globin gene expression in transgenic mice (11). Both the (3LCR and the aPRE are characterized by erythroid specific DNase 1 hypersensitive sites present at all stages of erytbroid development. In the j3LCR these DNase hypersensitive sites are located between 5-20 kb upstream of the human (3globin locus while in the aPRE, these sites are located between 30-40 kb upstream of the ca-globin gene cluster. Hypersensitive sites from both the ,B-LCR and the aPRE contain binding sites for the erythroid specific factors GATA-l and NF-E2 (16-18). In addition, hypersensitive site 2 (HS 2) of the j3LCR and the aPRE can act as erythroid specific transcriptional enhancers in transient transfection assays (19,20). Transgenic mice studies have demonstrated that the (3LCR can direct high levels of developmentally correct human r-globin gene expression (21,22). However, the ability of the aPRE to direct high levels of t-globin gene expression in transgenic mice has not been reported. In addition, the location of sequences within the r-globin gene responsible for its pattern of tissue and developmental specific expression is unknown. In this paper we demonstrate a simple assay system that allows us to study the tissue and developmental specific activation of the t-globin promoter in rnsgenic mice. We hoped that by linking regulatory sequences to the reporter gene ,B-galactosidase (lacZ), we would be able to analyze the tissue specificity and the developmental profile of expression relatively rapidly using classical histological techniques. The added advantage of staining sections is that the specificity of expression can be analyzed at the level of single cells. This is particularly important during early embryonic development. A construct containing 550 bp of 5' flanking sequencs from the human r-globin gene was fused to a lacZ reporter gene and linked to the aPRE. We show that the number of live born transgenic mice produced from this construct (r-promoter/ lacZ/aPRE) is significantly higher than the number produced
-40
-20
0
20
40
E
-4-B
SDK oligo
SV40 poly A
V
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v1
W3 HA3
-s I Pstl
a2 al
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EcoR5
poly linker
KpnlI ElI BH Sst
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C I Kpn 1
5Ih1 I Pst
LacZ
MATERIALS AND METHODS r-promoter/lacZ expression construct An Eco RI-Sty I restriction fragment containing 550 bp of r-globin 5' flanking sequences (cap site = 0) was isolated from the vector pCATEZ (23). The fragment was blunt ended and inserted into the blunt ended Hind III cut site of the vector pSDKlacZpa (gift of Dr. J.Rossant, Mount Sinai Research Institute, Toronto) (Figure lB). A 4.0 kb Bam HI fragment containing the xPRE (gift of Dr. D.Higgs, Institute of Molecular Medicine, Oxford) was then ligated into the Bam HI site of this vector 3' of the lacZ gene to create the vector r-promoter/ lacZ/oePRE (Figure 1C). The vector r-promoter/lacZ/ctPRE was cut with Asp 718 to release a 7.8 kb fragment containing rpromoter/lacZ/aPRE construct that was used for microinjection.
r geobin/cvPRE The 2.3 kb Eco RI-Bam HI fragment containing the human t-globin gene (from 550 bp 5' of the cap site to just beyond the poly A site) was linked to the aPRE (a 4.0 kb Bam HI fragment dicussed above) and cloned into Bluescript. The linearized 6.3 kb insert was then employed for microinjection.
r globinIi/LCR The 2.3 kb Eco RI-Bam HI fragment as indicated above, was linked to a 2.5 kb Bst EII-Cla I fragment containing HS 1 and HS 2 from the (3LCR (isolated from plasmid GSE 1417) (a gift from F.Grosveld) and cloned into Bluescript. The entire 4.8 kb insert was linearized and used for microinjection.
Generation of transgenic mice Linearized DNA fragments were isolated from agarose gels and purified on a sephadex-50 column, diluted with injection buffer to a final concentration of 2.5 pg/ml and passed through a 0.2 pm filter. Approximately 2-5 pl of the filtered DNA was injetd into the male pro-nucleus of fertilized eggs from adult P.O (Pathology, Outbred) mice. The injected eggs were maintained for several hours in M16 buffer at 37°C in a 5% C% incubator and transferred into the oviducts of pseudopregnant P.O. foster mothers.
A -60
when the construct contains the entire r-globin gene. We further show that the aPRE can direct correct temporal expression of the human r-globin promoter in a tissue specific manner in transgenic mice. These results suggest that sequences present in 550 bp's of r-globin 5' flanking region are sufficient to direct it's correct tissue and developmental specific pattern of expression.
s St1I a PREo -
Figue 1.Structure of the plasmid t-promoter/lacZi /aPRE. A. Line diagram of the human a-globin gene locus showing the positioniof the cPRE and erythroid specific hypersensitive site 40 (E). B. Expression Iplasmid pSDKIacZpa. SDK oligo contains Shine-Delgarno and Kozac sequenc es. C. lacZ/aPRE. This plasmid contains a 4.0 kb caPRE fra pment and 550 bp's of human t-globin 5' flaning region cloned into the Bam HI and Hind HI site of the plasmid pSDKlacZpa, respectively. H3 (Hind Im), SI (Sst l), IBHl (Bam HI), El (Eco
Plasimid t-promoter/ RM).
DNA analysis and estimation of gene copy number To identify transgenic founder mice, tail DNA from 3-4 week old pups was digested with one or two restriction enzymes and subjected to Southern blot analysis using either (1) a 1.8 kb Sac I fragment from the human #,rl gene for mice produced with the r-globin/cPRE or r-globin/I(LCR series or (2) a 2 kb Eco RI-Eco RV lacZ gene fragment for mice produced in the r-promoter/lacZ/atPRE series. Transgenic founder mice were mated and pups screened in the same manner to identify transgenic lines. To estinate gene copy number in transgenic lines from the positive mice, a DNA standard frmpstv equivalent to 2-500 copies of human aPRE per haploid genome and 5 pg of DNA from each transgenic line was cut with Bam
t-promoter/lacZ/cvPRE
Nucleic Acids Research, Vol. 20, No. 21 5657 HI and subjected to Southeren blot analysis employing a 4.0 kb Bam HI aPRE fragment as a probe. The autoradiograph was scanned by a LKB Ultrascan laser scanner to determine relative intensities of the human aPRE signals
effects in mammalian cells. In Table 1 we have calculated the efficiency of production of transgenic mice using three different constructs: two containing the entire human t-globin coding sequence (¢-globin/aPRE and t-globin/flLCR) and one containing the r-globin promoter linked to fl-galactosidase and the aPRE (r-promoter/lacZ/aPRE). We found that 12% of mice generated from eggs injected with r-promoter/lacZ/aPRE were transgenic compared with only 2.0% and 2.5% of mice generated
Histochemical analysis of 3-gal expression in transgenic embryos Transgenic males from each line were mated to female wild type P.O. mice. The appearance of a vaginal copulation plug was considered day 0.5 post-fertilization. 8.5-15 day old embryos in yolk sac were dissected free of decidua and fixed at 4°C in a solution containing: 0.2% gluteraldehyde, 0. IM phosphate buffer (pH 7.3), 2 mM MgCl2 and 5 mM Ethylene Glycol-bis N,N,N'-Tetraacetic Acid (EGTA). Embryos were then washed 3 x for 20 minutes at room temperature in buffer composed of 0.1M phosphate buffer (pH 7.3), 2 mM MgC12, 0.1% soduim desoxycholate, 0.02% Nonidet P-40 and 0.05% BSA. The embryos were transferred to a solution containing lmg/ml 5-Bromo-4-chloro-3-indoyl-,B-D-galactopyranoside (X-Gal), 5 mM of potassium ferro and ferricyanide and 0.25 mg/ml spermidine followed by incubation for 15 minutes to overnight at 37°C in total darkness. Whole embryos were then photographed on a dissection microscope. For more detailed histocemical analysis of (-gal activity, X-gal stained embryos were embedded in paraffin, sectioned (5Lm) and counterstained with eosin.
B
Quantitative analysis of fl-gal activity in transgenic embryos Individual 10.5 day old embryos in their yolk sac were dissected free of decidua and placenta and transferred to 1.5 ml eppendorf tubes. 100 1l of 250 mM Tris[hydroxmethyljaminomethane (Tris) pH. 7.5 was added to each tube and the embryos sonicated on ice. The lysates were spun on a microfuge for 10 minutes and the supernatant retained for (3-gal analysis. Protein concentration of each embryo lysate was then determined according to Bradford (24). fl-gal analysis was peformed on 40-70 yl of each embryonic lysate according to Herbomel et al. (25). f-gal activity of embryonic lysates in terms of mUnits/mg of protein was determined by comparing fl-gal activity of lysates to that of a f-galactosidase standard containing a known amount of f-gal activity.
C
RESULTS Increased number of transgenic lines generated with t-promoter/lacZ compared to t-globin DNA High concentrations of human globin protein may produce globin chain imbalance and result in death of transgenic mice embryos in utero (14). For this reason we decided to replace the t-globin coding sequence with the reporter gene f-galactosidase. High levels of f-galactosidase are not known to have any deleterious
A
A
; BV
Ys V
1I
I4
Table 1. Relative efficiency of transgenic mouse production
Construct
Pups tested
Positives
%
t-promoter/lacZ/ctPRE t-globin/uPRE
68 80 109
8 2 2
12.0 2.5 2.0
t-globin/OLCR
The number of live-born transgenic mice obtained with various globin LCR or aPRE constructs. The percentage calculated reflects the percentage of those tested that were positive for the transgene.
Figure 2. Tissue distribution of lacZ expression during development. X-gal staining of whole embryos carying the t-promoter/lacZ/aPRE construct is shown at day 8.5 (A) and 10.5 (B). Panel C shows a S Am section from a 10.5 day old embryo embedded in paraffin and counterstained with eosin. MD-maternal decidua, YSyolk sac, BV-blood vessel, V-ventricle, A-atrium. The magnification factor for panel A is 30:1, for panel B is 25:1 and for panel C is 140:1.
5658 Nucleic Acids Research, Vol. 20, No. 21 from eggs injected with the entire t-globin gene linked to the j3LCR and aPRE, respectively. In other words, by replacing the r-globin coding sequence withfi-galactosidase we could produce transgenic lines 5-6 times as efficiently. The reduced number of live born transgenics containing the r-globin coding sequence suggests that death of positive embryos, possibly due to thalassaemia, may be occurring.
The caPRE can direct correct tissue and developmental expression of the t-promoter/lacZ gene in transgenic mice In the developing mouse, the first site of erythropoiesis occurs in yolk sac blood island from between 8-14 days of gestation. The major site of erythropoesis then shifts to the liver. Whitelaw et al. (26) showed that mouse t-globin expression reached its peak at about 9.5 days of development in the yolk sac and disappeared by about 15-16 days of development, coincident with the shift of erythropoesis to the liver. We therefore wished to determine whether the cPRE could direct a similar temporal and tissue specific pattern of human r-promoter/lacZ expression in transgenic mice. The results of X-gal staining oftransgenic embryos can be seen in Figure 2. X-gal staining of embyros from transgenic lines (1-5) revealed that lacZ gene activity was first evident at 8.5 days of development and expression was restricted to the embryonic yolk sac. At this stage the staining is in fact, limited to the blood islands that form a ring around the yolk sac (Figure 2A). A similar embryo stained with X-gal, embedded in paraffin and sectioned revealed that fi-galactosidase expression is restricted to the primitive erythroid cells developing in the visceral endoderm of the yolk sac (data not shown). Figure 2B shows a stained whole-mount at day 10.5. Again, only the erythroid cells in the blood vessels of the yolk sack are blue. At this stage, vascularization over the entire surface of the yolk sac has been completed. After sectioning, we can see that staining is limited to the erythroid cells in the yolk sac, the heart and various blood vessels (see Figure 2C). In all lines, staining was greatest at days 8.5 and 9.5. By day 12.5, as definitive erythroid cells produced by the liver start to make a significant contribution to the total number of red cells, the number of erythroid cells that stain blue steadily declined. Also, the intensity of the stain within a single cell declined. No staining was found to occur in the fetal liver at any stage of its development. It was theoretically possible that lack of (3galactosidase activity in the liver was due to poor diffusion of X-gal into the liver. We therefore removed livers from some litters and stained these directly in X-gal. No fi-galactosidase activity was observed. By day 15, no stained cells could be detected in the yolk sac or in the embryo proper. These data suggest that correct tissue and developmental expression of the construct is occurring. t-promoter/lacZ gene expression in transgenic mice is not positively correlated with copy number Of the 8 transgenic mice that were positive for the ¢-promoter/ lacZ/atPRE transgene (see Table 1), 2 were found to be mosaic and were discontinued. The remaining 6 were all germline and one of these six lines showed no ,-galactosidase expression at any stage of development nor in any cell type. Although the temporal and tissue specific expression pattern of transgene in the remaining 5 transgenic lines was identical, the degree of X-gal staining between lines was variable: while
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:#
Figure 3. Gene copy number in the ¢-promoter/lacZ/aPRE lines. Estimates of gene copy number of each transgenic mouse line (1-5) compared to a set of pre-determined standards (2-500 copies of cPRE). 5 Ug of DNA from each mouse line was cut with Bam HI and probed with a radiolabled 4 kb caPRE fragment and a 280 bp mouse ca-globin fragment (internal control) (see Materials and Methods).
O 50 31
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T
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30'
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E 10,
1
2
3
4
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350
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12
30
Trenegenic Line copy number
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Figure 4. Quantitative analysis of j3-galactosidase activity from transgenic embryos. Data represents average fl-galactosidase activity in 10-15 transgenic embryos from transgenic lines I -5. Copy number of t-promoter/lacZ/cVPRE is indicated. Error bars indicate standard deviation.
embryos from some transgenic lines required only 5-15 minutes of incubation in X-gal solution to visualize (3-gal activity, embryos from other transgenic lines took hours or even overnight incubation for fl-gal activity to be detected. It was possible that variability in lacZ gene expression was due to differences in transgene copy number. We therefore wished to determine the copy number of ¢-promoter/lacZ/aPRE in each transgenic line and relate this to a quantitative analysis of fl-gal activity. Employing the aPRE as a probe, Southern blot analysis demonstrated that transgene copy number varied from between 12 to 350 copies in the 5 transgenic lines (Figure 3). To obtain a quantitative analysis of lacZ gene activity, embryos from transgenic lines 1-5 were individually sonicated and the lysates were assayed for f-galactosidase activity. Data showed that while there was little variability of fl-gal activity from embryos within a line, substantial variability of fl-gal activity in embryos occurred between the transgenic lines (Figure 4). Lines 1 and 5 both contained approximately 12 copies of ¢-promoter/
lacZ/aePRE and showed the greatest level of fl-gal activity
(38-40 mUnits/mg) while line 3, containing approximately 30 copies of r-promoter/lacZ/aPRE had almost 13 times less activity (3.5 mUnits/mg of protein). Transgenic lines 2 and 4 contained approximately 250 and 350 copies of r-promoter/lacZ/cvPRE, respectively, yet showed f-gal activity of only 2.5 and 9
Nucleic Acids Research, Vol. 20, No. 21 5659 mUnits/mg of protein. These results suggest that t-promoter/lacZ expression in the presence of the ctPRE was not copy number dependent.
DISCUSSION We have demonstrated that a t-globin promoter/lacZ construct is more efficient at generating transgenic lines than an entire globin gene. Grosveld et al., (14) showed that high concentrations of human globin protein in mice may produce globin chain imbalance and result in death of transgenic embryos in utero. In addition, Albitar et al., (22) showed a lower efficiency in generating live born transgenic mice compared to transgenic embryos when employing a t- or ca-globin gene linked to the 3LCR. Our data demonstrated that 6 times as many live born transgenic mice lines were produced employing a t-promoter/ lacZ/aPRE construct compared to an entire t-globin gene linked to the atPRE or (3LCR. Although transgenic embryos containing the entire t-globin gene linked to the aPRE or ,BLCR were not examined for obvious signs of anaemia, we hypothesize that the reduced number of live born transgenic mice containing these constructs may be due to globin chain imbalance resulting in death of transgenic embryos in utero. Our results suggest that if one is studying the transcriptional regulation of a gene that may be lethal when over produced, linking the regulatory sequences to a neutral reporter gene can maximize the number of transgenic lines generated. Studies have shown that normal developmental control of the human t-globin gene linked to the (3LCR in transgenic mice was not dependent on the presence in cis of the human ca-globin gene (21,22). Down regulation of embryonic P-globin expression to fetal/adult ct-globin occurs between 12 and 16 days of gestation and parallels the down regulation of the corresponding endogenous mouse globin genes. We wished to develop a simple assay system that would enable us to determine the location of critical sequences responsible for r globin's pattern of tissue and developmental specific expression in transgenic mice. Based on simple histochemical analysis from individual transgenic embryos, our data shows that r-promoter/lacZ gene expression in the presence of the aPRE, parallels the known expression pattern of the endogenous mouse t-globin gene. This result suggests that sequences present in 550 bp of t-globin 5' flanking region are sufficient to direct correct tissue and developmental expression of the t-globin gene in transgenic mice. We have currently created a series of t-promoter/lacZ/aPRE constructs containing successively smaller amounts of t-globin 5' flanking region. By generating transgenic mice lines containing these constructs we hope to more carefully delineate these control sequences. Higgs et al., (11) showed that when the ca-globin gene was linked to the caPRE and transfected into MELCs, ca-globin expression showed some relationship between copy number and transcript levels. However, the relationship was not linear: there appeared to be a threshold level beyond which increasing copy number did not give rise to increased expression levels. Although our data shows the caPRE is able to direct correct tissue and developmental specificity of a t-promoter/lacZ construct in transgenic mice, such a a relationship between copy number and lacZ expression levels is not observed. Vyas et al. (27) has suggested that since the human a-globin cluster appears to be in a constitutively 'open' chromatin structure, the caPRE unlike
the ,B-LCR may not have a domain 'opening' function. This suggests that the lack of t-promoter/lacZ/aPRE copy dependent expression we have observed may be due to position effects. Although some workers have found that the ,BLCR (or HS 2 alone) does confer copy-dependent expression in transgenic mice (28,14,15) others have observed a poor correlation (9,29-31). It would clearly be interesting to replace the aPRE with the ,BLCR in the t-promoter/lacZ construct to determine if copy number dependent expression is observed. The assay system described in this report should facilitate a clearer understanding of the sequences responsible for the developmental and tissue specific regulation of human globin genes.
ACKNOWLEDGEMENTS We thank Dr. D.Higgs (Institute of Molecular Medicine, Oxford) for the atPRE fragment and Dr. J.Rossant (Mount Sinai Research Institute, Toronto) for the pSDKlacZpa clone. This work was supported by MRC project grant G9008512CB awarded to E.W. and MRC project grant G8822153CB to N.J.P. E.W. would like to thank Dr. David Weatherall for providing space for this work to be carried out.
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5660 Nucleic Acids Research, Vol. 20, No. 21 26. Whitelaw, E., Tsai, S.F., Hogben, P. and Orkin, S.H. (1990) Mol. Cell. Biol. 10, 6596-6606. 27. Vyas, P., Vickers, M.A., Simmons, D.L., Ayyub, H., Craddock, C.F. and Higgs, D.R. (1992) Cell 69, 781-793. 28. Fraser, P.J., Hurst, S., Collis, P. and Grosveld, F. (1990) Nucl. Acids Res. 18, 352-355. 29. Curtin, P.T., Liu, D., Liu, W., Chang, J.C. and Kan, Y.W. (1989) Proc. Natl. Acad. Sci. USA 86, 5439-5443. 30. Forrester, W.C., Novak, U., Gelinas, R. and Groudine, M. (1989) Proc. Nail. Acad. Sci. USA 86, 5439-5443. 31. Morley, B.J., Abbot, C.A. Sharpe, J.A., Lida, J., Chan-Thomas, P.S. and Wood, W.G. (1992) Mol. Cell Biol. 12, 2057-2066.
Nucleic Acids Research, Vol. 20, No. 21 5655 -5660
The developmental regulation of the human -globin in transgenic mice employing j3-galactosidase as a
gene
reporter gene
M.D.Pondel, N.J.Proudfoot, C.Whitelaw and E.Whitelawl,* Sir William Dunn School of Pathology, Oxford University, Oxford OX1 3RE, UK and 1Department of Biochemistry, University of Sydney, Sydney, NSW 2006, Australia Received August 5, 1992; Accepted September 25, 1992
ABSTRACT We have investigated the developmental and tissue specific expression of the human embryonic t-globin gene in transgenic mice. A construct containing 550 bp of t-globin 5' flanking region, fused to a ,Bgalactosidase (lacZ) reporter gene and linked to the locus control region (LCR)-like a positive regulatory element (aPRE) was employed for the production of transgenic mice. Firstly, we compared the number of live born transgenic mice containing this construct to the number of live born transgenic mice containing the entire t-globin gene linked to the aPRE or the ,3LCR. Data showed that 12% of mice generated from eggs injected with P-promoter/lacZ/aPRE DNA were transgenic compared to only 2% of mice generated from eggs injected with the entire t-globin gene linked to the aPRE or the ,BLCR. The reduced number of live born transgenic mice containing the latter constructs suggests that death of transgenic embryos, posssibly due to thalassaemia, may be occurring. X-gal staining of whole embryos containing the lacZ gene revealed that t-globin promoter activity was most pronounced at 8.5 - 9.5 days of development and was restricted to erythroid cells. By 15 days of development, no t-globin promoter activity was detected. These results suggest that the aPRE can direct high level expression from the t-globin promoter and that sequences required for the correct tissue and developmental specific expression of the human f-globin gene are present within 550 bp's of 5' flanking region. Sequences within the body of the P-globin gene or 3' of the cap site do not appear to be necessary for correct P-globin developmental regulation. INTRODUCTION The hemoglobin molecule is a tetrameric protein containing two a like globin chains and two ,B like globin chains. The human cx-globin gene cluster consists of three fuctional genes arranged 5'-v2-a2-al -3' at the tip of chromosome 16p. The human ,*
To whom correspondence should be addressed
globin cluster consists of five functional genes arranged 5-e-GyA,y-6-3-3' on the tip of chromosome lp. The genes within each cluster are regulated in a tissue and developmental stage specific manner to produce embryonic (r2E2, a2E2 and t2-y2), fetal (a2,y2) and adult (a2a2 and a2j32) hemoglobin. In the human ,B cluster, the embryonic E-globin gene is expressed from about 3 to7 weeks of development followed by a switch to expression of the two adjacent fetal 'y-globin genes. An additional switch from -y-globin expression to f-globin gene expression occurs at birth (1). In the ca cluster, a single switch from t2 to c2 and a l expression takes place at 6-7 weeks of development (2). The mechanism(s) responsible for the coordinated regulation of these two physically separated gene loci is hypothesized to occur at the transcriptional level (3). Transgenic mice have provided a useful system for the study of human globin gene regulation. When a 4 kb fragment containing the entire 3-globin gene is introduced into the genome of mice, correct developmental and tissue specific expression is attained. Expression, however, is less than 5% of endogenous globin expression and dependent upon the site of integration in the genome (4-6). Transgenic mice containing a fragment that includes the entire human a-globin gene show no expression of the introduced gene (7-9). When the ax- and ,3-globin genes on intact chromosomes 16 and 11 are transferred to human X mouse erythroleukemia cells (MELCs), their level of expression is similar to that of the endogenous mouse globin genes (10-13). These results suggest that additional cis acting regulatory sequences remote from the a- and 0-globin gene cluster are necessary to achieve high levels of position independent a and j-globin expression in transgenic mice. Studies have shown that high levels of human ,B-globin gene expression in stably transfected cell lines and transgenic mice is dependent on DNA sequences located 5-20 kb upstream of the human j3-globin locus. These sequences, collectively referred to as the locus control region (LCR) can direct high level expression of both at- and ,B-globin genes in a copy dependent, position independent manner in transgenic mice (7,9,14,15). More recently, an LCR like a-globin positive regulatory element (aPRE) located 30-40 kb upstream the human t-globin gene
5656 Nucleic Acids Research, Vol. 20, No. 21 has been identified. Like the ,BLCR, the aPRE is capable of directing high levels of a-globin gene expression in transgenic mice (11). Both the (3LCR and the aPRE are characterized by erythroid specific DNase 1 hypersensitive sites present at all stages of erytbroid development. In the j3LCR these DNase hypersensitive sites are located between 5-20 kb upstream of the human (3globin locus while in the aPRE, these sites are located between 30-40 kb upstream of the ca-globin gene cluster. Hypersensitive sites from both the ,B-LCR and the aPRE contain binding sites for the erythroid specific factors GATA-l and NF-E2 (16-18). In addition, hypersensitive site 2 (HS 2) of the j3LCR and the aPRE can act as erythroid specific transcriptional enhancers in transient transfection assays (19,20). Transgenic mice studies have demonstrated that the (3LCR can direct high levels of developmentally correct human r-globin gene expression (21,22). However, the ability of the aPRE to direct high levels of t-globin gene expression in transgenic mice has not been reported. In addition, the location of sequences within the r-globin gene responsible for its pattern of tissue and developmental specific expression is unknown. In this paper we demonstrate a simple assay system that allows us to study the tissue and developmental specific activation of the t-globin promoter in rnsgenic mice. We hoped that by linking regulatory sequences to the reporter gene ,B-galactosidase (lacZ), we would be able to analyze the tissue specificity and the developmental profile of expression relatively rapidly using classical histological techniques. The added advantage of staining sections is that the specificity of expression can be analyzed at the level of single cells. This is particularly important during early embryonic development. A construct containing 550 bp of 5' flanking sequencs from the human r-globin gene was fused to a lacZ reporter gene and linked to the aPRE. We show that the number of live born transgenic mice produced from this construct (r-promoter/ lacZ/aPRE) is significantly higher than the number produced
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MATERIALS AND METHODS r-promoter/lacZ expression construct An Eco RI-Sty I restriction fragment containing 550 bp of r-globin 5' flanking sequences (cap site = 0) was isolated from the vector pCATEZ (23). The fragment was blunt ended and inserted into the blunt ended Hind III cut site of the vector pSDKlacZpa (gift of Dr. J.Rossant, Mount Sinai Research Institute, Toronto) (Figure lB). A 4.0 kb Bam HI fragment containing the xPRE (gift of Dr. D.Higgs, Institute of Molecular Medicine, Oxford) was then ligated into the Bam HI site of this vector 3' of the lacZ gene to create the vector r-promoter/ lacZ/oePRE (Figure 1C). The vector r-promoter/lacZ/ctPRE was cut with Asp 718 to release a 7.8 kb fragment containing rpromoter/lacZ/aPRE construct that was used for microinjection.
r geobin/cvPRE The 2.3 kb Eco RI-Bam HI fragment containing the human t-globin gene (from 550 bp 5' of the cap site to just beyond the poly A site) was linked to the aPRE (a 4.0 kb Bam HI fragment dicussed above) and cloned into Bluescript. The linearized 6.3 kb insert was then employed for microinjection.
r globinIi/LCR The 2.3 kb Eco RI-Bam HI fragment as indicated above, was linked to a 2.5 kb Bst EII-Cla I fragment containing HS 1 and HS 2 from the (3LCR (isolated from plasmid GSE 1417) (a gift from F.Grosveld) and cloned into Bluescript. The entire 4.8 kb insert was linearized and used for microinjection.
Generation of transgenic mice Linearized DNA fragments were isolated from agarose gels and purified on a sephadex-50 column, diluted with injection buffer to a final concentration of 2.5 pg/ml and passed through a 0.2 pm filter. Approximately 2-5 pl of the filtered DNA was injetd into the male pro-nucleus of fertilized eggs from adult P.O (Pathology, Outbred) mice. The injected eggs were maintained for several hours in M16 buffer at 37°C in a 5% C% incubator and transferred into the oviducts of pseudopregnant P.O. foster mothers.
A -60
when the construct contains the entire r-globin gene. We further show that the aPRE can direct correct temporal expression of the human r-globin promoter in a tissue specific manner in transgenic mice. These results suggest that sequences present in 550 bp's of r-globin 5' flanking region are sufficient to direct it's correct tissue and developmental specific pattern of expression.
s St1I a PREo -
Figue 1.Structure of the plasmid t-promoter/lacZi /aPRE. A. Line diagram of the human a-globin gene locus showing the positioniof the cPRE and erythroid specific hypersensitive site 40 (E). B. Expression Iplasmid pSDKIacZpa. SDK oligo contains Shine-Delgarno and Kozac sequenc es. C. lacZ/aPRE. This plasmid contains a 4.0 kb caPRE fra pment and 550 bp's of human t-globin 5' flaning region cloned into the Bam HI and Hind HI site of the plasmid pSDKlacZpa, respectively. H3 (Hind Im), SI (Sst l), IBHl (Bam HI), El (Eco
Plasimid t-promoter/ RM).
DNA analysis and estimation of gene copy number To identify transgenic founder mice, tail DNA from 3-4 week old pups was digested with one or two restriction enzymes and subjected to Southern blot analysis using either (1) a 1.8 kb Sac I fragment from the human #,rl gene for mice produced with the r-globin/cPRE or r-globin/I(LCR series or (2) a 2 kb Eco RI-Eco RV lacZ gene fragment for mice produced in the r-promoter/lacZ/atPRE series. Transgenic founder mice were mated and pups screened in the same manner to identify transgenic lines. To estinate gene copy number in transgenic lines from the positive mice, a DNA standard frmpstv equivalent to 2-500 copies of human aPRE per haploid genome and 5 pg of DNA from each transgenic line was cut with Bam
t-promoter/lacZ/cvPRE
Nucleic Acids Research, Vol. 20, No. 21 5657 HI and subjected to Southeren blot analysis employing a 4.0 kb Bam HI aPRE fragment as a probe. The autoradiograph was scanned by a LKB Ultrascan laser scanner to determine relative intensities of the human aPRE signals
effects in mammalian cells. In Table 1 we have calculated the efficiency of production of transgenic mice using three different constructs: two containing the entire human t-globin coding sequence (¢-globin/aPRE and t-globin/flLCR) and one containing the r-globin promoter linked to fl-galactosidase and the aPRE (r-promoter/lacZ/aPRE). We found that 12% of mice generated from eggs injected with r-promoter/lacZ/aPRE were transgenic compared with only 2.0% and 2.5% of mice generated
Histochemical analysis of 3-gal expression in transgenic embryos Transgenic males from each line were mated to female wild type P.O. mice. The appearance of a vaginal copulation plug was considered day 0.5 post-fertilization. 8.5-15 day old embryos in yolk sac were dissected free of decidua and fixed at 4°C in a solution containing: 0.2% gluteraldehyde, 0. IM phosphate buffer (pH 7.3), 2 mM MgCl2 and 5 mM Ethylene Glycol-bis N,N,N'-Tetraacetic Acid (EGTA). Embryos were then washed 3 x for 20 minutes at room temperature in buffer composed of 0.1M phosphate buffer (pH 7.3), 2 mM MgC12, 0.1% soduim desoxycholate, 0.02% Nonidet P-40 and 0.05% BSA. The embryos were transferred to a solution containing lmg/ml 5-Bromo-4-chloro-3-indoyl-,B-D-galactopyranoside (X-Gal), 5 mM of potassium ferro and ferricyanide and 0.25 mg/ml spermidine followed by incubation for 15 minutes to overnight at 37°C in total darkness. Whole embryos were then photographed on a dissection microscope. For more detailed histocemical analysis of (-gal activity, X-gal stained embryos were embedded in paraffin, sectioned (5Lm) and counterstained with eosin.
B
Quantitative analysis of fl-gal activity in transgenic embryos Individual 10.5 day old embryos in their yolk sac were dissected free of decidua and placenta and transferred to 1.5 ml eppendorf tubes. 100 1l of 250 mM Tris[hydroxmethyljaminomethane (Tris) pH. 7.5 was added to each tube and the embryos sonicated on ice. The lysates were spun on a microfuge for 10 minutes and the supernatant retained for (3-gal analysis. Protein concentration of each embryo lysate was then determined according to Bradford (24). fl-gal analysis was peformed on 40-70 yl of each embryonic lysate according to Herbomel et al. (25). f-gal activity of embryonic lysates in terms of mUnits/mg of protein was determined by comparing fl-gal activity of lysates to that of a f-galactosidase standard containing a known amount of f-gal activity.
C
RESULTS Increased number of transgenic lines generated with t-promoter/lacZ compared to t-globin DNA High concentrations of human globin protein may produce globin chain imbalance and result in death of transgenic mice embryos in utero (14). For this reason we decided to replace the t-globin coding sequence with the reporter gene f-galactosidase. High levels of f-galactosidase are not known to have any deleterious
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Table 1. Relative efficiency of transgenic mouse production
Construct
Pups tested
Positives
%
t-promoter/lacZ/ctPRE t-globin/uPRE
68 80 109
8 2 2
12.0 2.5 2.0
t-globin/OLCR
The number of live-born transgenic mice obtained with various globin LCR or aPRE constructs. The percentage calculated reflects the percentage of those tested that were positive for the transgene.
Figure 2. Tissue distribution of lacZ expression during development. X-gal staining of whole embryos carying the t-promoter/lacZ/aPRE construct is shown at day 8.5 (A) and 10.5 (B). Panel C shows a S Am section from a 10.5 day old embryo embedded in paraffin and counterstained with eosin. MD-maternal decidua, YSyolk sac, BV-blood vessel, V-ventricle, A-atrium. The magnification factor for panel A is 30:1, for panel B is 25:1 and for panel C is 140:1.
5658 Nucleic Acids Research, Vol. 20, No. 21 from eggs injected with the entire t-globin gene linked to the j3LCR and aPRE, respectively. In other words, by replacing the r-globin coding sequence withfi-galactosidase we could produce transgenic lines 5-6 times as efficiently. The reduced number of live born transgenics containing the r-globin coding sequence suggests that death of positive embryos, possibly due to thalassaemia, may be occurring.
The caPRE can direct correct tissue and developmental expression of the t-promoter/lacZ gene in transgenic mice In the developing mouse, the first site of erythropoiesis occurs in yolk sac blood island from between 8-14 days of gestation. The major site of erythropoesis then shifts to the liver. Whitelaw et al. (26) showed that mouse t-globin expression reached its peak at about 9.5 days of development in the yolk sac and disappeared by about 15-16 days of development, coincident with the shift of erythropoesis to the liver. We therefore wished to determine whether the cPRE could direct a similar temporal and tissue specific pattern of human r-promoter/lacZ expression in transgenic mice. The results of X-gal staining oftransgenic embryos can be seen in Figure 2. X-gal staining of embyros from transgenic lines (1-5) revealed that lacZ gene activity was first evident at 8.5 days of development and expression was restricted to the embryonic yolk sac. At this stage the staining is in fact, limited to the blood islands that form a ring around the yolk sac (Figure 2A). A similar embryo stained with X-gal, embedded in paraffin and sectioned revealed that fi-galactosidase expression is restricted to the primitive erythroid cells developing in the visceral endoderm of the yolk sac (data not shown). Figure 2B shows a stained whole-mount at day 10.5. Again, only the erythroid cells in the blood vessels of the yolk sack are blue. At this stage, vascularization over the entire surface of the yolk sac has been completed. After sectioning, we can see that staining is limited to the erythroid cells in the yolk sac, the heart and various blood vessels (see Figure 2C). In all lines, staining was greatest at days 8.5 and 9.5. By day 12.5, as definitive erythroid cells produced by the liver start to make a significant contribution to the total number of red cells, the number of erythroid cells that stain blue steadily declined. Also, the intensity of the stain within a single cell declined. No staining was found to occur in the fetal liver at any stage of its development. It was theoretically possible that lack of (3galactosidase activity in the liver was due to poor diffusion of X-gal into the liver. We therefore removed livers from some litters and stained these directly in X-gal. No fi-galactosidase activity was observed. By day 15, no stained cells could be detected in the yolk sac or in the embryo proper. These data suggest that correct tissue and developmental expression of the construct is occurring. t-promoter/lacZ gene expression in transgenic mice is not positively correlated with copy number Of the 8 transgenic mice that were positive for the ¢-promoter/ lacZ/atPRE transgene (see Table 1), 2 were found to be mosaic and were discontinued. The remaining 6 were all germline and one of these six lines showed no ,-galactosidase expression at any stage of development nor in any cell type. Although the temporal and tissue specific expression pattern of transgene in the remaining 5 transgenic lines was identical, the degree of X-gal staining between lines was variable: while
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Figure 3. Gene copy number in the ¢-promoter/lacZ/aPRE lines. Estimates of gene copy number of each transgenic mouse line (1-5) compared to a set of pre-determined standards (2-500 copies of cPRE). 5 Ug of DNA from each mouse line was cut with Bam HI and probed with a radiolabled 4 kb caPRE fragment and a 280 bp mouse ca-globin fragment (internal control) (see Materials and Methods).
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Figure 4. Quantitative analysis of j3-galactosidase activity from transgenic embryos. Data represents average fl-galactosidase activity in 10-15 transgenic embryos from transgenic lines I -5. Copy number of t-promoter/lacZ/cVPRE is indicated. Error bars indicate standard deviation.
embryos from some transgenic lines required only 5-15 minutes of incubation in X-gal solution to visualize (3-gal activity, embryos from other transgenic lines took hours or even overnight incubation for fl-gal activity to be detected. It was possible that variability in lacZ gene expression was due to differences in transgene copy number. We therefore wished to determine the copy number of ¢-promoter/lacZ/aPRE in each transgenic line and relate this to a quantitative analysis of fl-gal activity. Employing the aPRE as a probe, Southern blot analysis demonstrated that transgene copy number varied from between 12 to 350 copies in the 5 transgenic lines (Figure 3). To obtain a quantitative analysis of lacZ gene activity, embryos from transgenic lines 1-5 were individually sonicated and the lysates were assayed for f-galactosidase activity. Data showed that while there was little variability of fl-gal activity from embryos within a line, substantial variability of fl-gal activity in embryos occurred between the transgenic lines (Figure 4). Lines 1 and 5 both contained approximately 12 copies of ¢-promoter/
lacZ/aePRE and showed the greatest level of fl-gal activity
(38-40 mUnits/mg) while line 3, containing approximately 30 copies of r-promoter/lacZ/aPRE had almost 13 times less activity (3.5 mUnits/mg of protein). Transgenic lines 2 and 4 contained approximately 250 and 350 copies of r-promoter/lacZ/cvPRE, respectively, yet showed f-gal activity of only 2.5 and 9
Nucleic Acids Research, Vol. 20, No. 21 5659 mUnits/mg of protein. These results suggest that t-promoter/lacZ expression in the presence of the ctPRE was not copy number dependent.
DISCUSSION We have demonstrated that a t-globin promoter/lacZ construct is more efficient at generating transgenic lines than an entire globin gene. Grosveld et al., (14) showed that high concentrations of human globin protein in mice may produce globin chain imbalance and result in death of transgenic embryos in utero. In addition, Albitar et al., (22) showed a lower efficiency in generating live born transgenic mice compared to transgenic embryos when employing a t- or ca-globin gene linked to the 3LCR. Our data demonstrated that 6 times as many live born transgenic mice lines were produced employing a t-promoter/ lacZ/aPRE construct compared to an entire t-globin gene linked to the atPRE or (3LCR. Although transgenic embryos containing the entire t-globin gene linked to the aPRE or ,BLCR were not examined for obvious signs of anaemia, we hypothesize that the reduced number of live born transgenic mice containing these constructs may be due to globin chain imbalance resulting in death of transgenic embryos in utero. Our results suggest that if one is studying the transcriptional regulation of a gene that may be lethal when over produced, linking the regulatory sequences to a neutral reporter gene can maximize the number of transgenic lines generated. Studies have shown that normal developmental control of the human t-globin gene linked to the (3LCR in transgenic mice was not dependent on the presence in cis of the human ca-globin gene (21,22). Down regulation of embryonic P-globin expression to fetal/adult ct-globin occurs between 12 and 16 days of gestation and parallels the down regulation of the corresponding endogenous mouse globin genes. We wished to develop a simple assay system that would enable us to determine the location of critical sequences responsible for r globin's pattern of tissue and developmental specific expression in transgenic mice. Based on simple histochemical analysis from individual transgenic embryos, our data shows that r-promoter/lacZ gene expression in the presence of the aPRE, parallels the known expression pattern of the endogenous mouse t-globin gene. This result suggests that sequences present in 550 bp of t-globin 5' flanking region are sufficient to direct correct tissue and developmental expression of the t-globin gene in transgenic mice. We have currently created a series of t-promoter/lacZ/aPRE constructs containing successively smaller amounts of t-globin 5' flanking region. By generating transgenic mice lines containing these constructs we hope to more carefully delineate these control sequences. Higgs et al., (11) showed that when the ca-globin gene was linked to the caPRE and transfected into MELCs, ca-globin expression showed some relationship between copy number and transcript levels. However, the relationship was not linear: there appeared to be a threshold level beyond which increasing copy number did not give rise to increased expression levels. Although our data shows the caPRE is able to direct correct tissue and developmental specificity of a t-promoter/lacZ construct in transgenic mice, such a a relationship between copy number and lacZ expression levels is not observed. Vyas et al. (27) has suggested that since the human a-globin cluster appears to be in a constitutively 'open' chromatin structure, the caPRE unlike
the ,B-LCR may not have a domain 'opening' function. This suggests that the lack of t-promoter/lacZ/aPRE copy dependent expression we have observed may be due to position effects. Although some workers have found that the ,BLCR (or HS 2 alone) does confer copy-dependent expression in transgenic mice (28,14,15) others have observed a poor correlation (9,29-31). It would clearly be interesting to replace the aPRE with the ,BLCR in the t-promoter/lacZ construct to determine if copy number dependent expression is observed. The assay system described in this report should facilitate a clearer understanding of the sequences responsible for the developmental and tissue specific regulation of human globin genes.
ACKNOWLEDGEMENTS We thank Dr. D.Higgs (Institute of Molecular Medicine, Oxford) for the atPRE fragment and Dr. J.Rossant (Mount Sinai Research Institute, Toronto) for the pSDKlacZpa clone. This work was supported by MRC project grant G9008512CB awarded to E.W. and MRC project grant G8822153CB to N.J.P. E.W. would like to thank Dr. David Weatherall for providing space for this work to be carried out.
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