ee gy ee ee my ee
`
`
`
`Figure 4. Primer extension analysis of fetal liver RNA from
`HS I-VI B transgenic mice. Human a-, mouse a-, and mouse
`B-globin-specific oligonucleotides were end labeled with
`[a-2P|ATP (3000 Ci/mm) and hybridized together with 5 yg of
`mouse fetal liver RNA or 0.5 ng of human reticulocyte RNA
`and then extended with reverse transcriptase to map the 5' ends
`of human B-, mouse a-, and mouse B-globin mRNAs. The
`products were electrophoresed on an 8.0% urea—polyacryl-
`amide gel, and the gel was autoradiographed for 8 hr at — 70°C
`with an intensifying screen. The authentic human B-globin
`primerextension productis 98 bp, and the correct mouse a- and
`B-globin products are 65 and 53 bp,respectively. Markers are
`end-labeled Hpail fragments of the plasmid pSP64. Accurate
`quantitative values of human §-globin and mouse £-globin
`mRNAs were determined by solution hybridization with
`human f-globin and mouse £-globin-specific oligonucleotides
`as described by Townesetal. (1985b]. Levels of human f-globin
`mRNA expressed as a percentage of endogenous mouse B-
`globin mRNAare listed in parenthesis after each sample
`number.
`
`B-Globin gene expression in transgenic mice
`
`{30} B construct. Levels of human @-globin mRNA
`ranged from 18 to 316% of endogenous mouse B-globin
`mRNA.Whenthesevalues were corrected for transgene
`copy number, the average level of expression per gene
`copy was 108% of endogenous mouse B-globin mRNA
`(Table 1).
`A construct that containedall five upstream HSsites
`on a smaller fragment (22 kb} was also assayed for ac-
`tivity. Nine animals containing intact copies of the HS
`I-V (22) B transgene(Fig. 2} were obtained, and all nine
`expressed human #-globin mRNAin fetal liver. Fetal
`liver RNA from eight of these samples was analyzed by
`primerextension. Theresults are illustrated in Figure 6.
`All eight animals expressed correctly initiated human p-
`globin mRNA,andthelevels of expression ranged from
`52 to 380% of endogenous mouse B-globin mRNA. The
`lowest expressor (4854), which expressed human f-
`globin mRNA at 1.0% of the level of mouse B-globin
`mRNA,was not included on the gel. When the level of
`expression for al] nine animals was corrected for trans-
`gene copy number, the average level of expression per
`gene copy was 109% of endogenous mouse B-globin
`mRNA(Table 1).
`To determine whether all five upstream HSsites are
`required for high level erythroid expression, a construct
`containing only HS I and HS II on a 13-kb Mlul—Clal
`fragment wasinserted upstream of the human £-globin
`gene (Fig. 2) and tested for activity. Thirteen animals
`that contained intact copies of the HS II (13) 6 trans-
`
`Table 1. Summary of HS f transgene expression
`Percent
`endogenous
`Fraction mouse B-globin
`expressors mRNA*
`
`Transgene
`
`Percent expression
`Per gene copy?
`mean
`range
`
`mousea- and B-globin products are 65 and 53 bp, respec-
`tively. All three of the animals that contained the HS
`I-VI B transgene expressed correctly initiated human §-
`20-84
`52
`5-26
`3/3
`HS I-VI p
`globin mRNA,andthelevels of expression; which are
`
`HSI-V(30)@=13/13 18-316 108 16-200
`
`
`listed in parentheses after each sample number, ranged
`HSI-V (22} 6
`9/9
`1-380
`109
`2-208
`from 5.0 to 26% of endogenous mouse B-globin mRNA.
`HS 1,01 (13) B
`13/13
`9-347
`49
`9-92
`HS Hf [5.8] B
`6/7
`8-108
`40
`6—84
`As there are four copies of the mouse B-globin gene per
`HS 0 [1.9] p
`4/4
`56-194
`40
`13-63
`diploid genome(28$ and 26*alleles in the B single haplo-
`B
`7/23
`0.2-23
`03
`0.1-0.6
`type mouse; Weaveret al. 1981), the levels of human and
`mouse f-globin mRNAsweredivided by their respective
`gene copy numbers to make a direct comparison of ex-
`pression. The corrected values for human {-globin
`mRNAranged from 20 to 84% of endogenous mouse B-
`globin mRNA,andthe average level of expression was
`52% per gene copy (Table 1).
`To determine whether the downstream HSVI site was
`required for high level human £-globin gene expression,
`a construct containing only the five upstream HSsites
`(HS 1-V (30) 8; Fig. 2] was analyzed in transgenic mice.
`This constrict contains the five HS sites on a 30-kb
`fragment linked upstream of the human £-globin gene.
`Thirteen animals that contained intact copies of the
`transgene were obtained, and all 13 expressed human p-
`globin mRNAin fetal
`liver. Figure 5 illustrates the
`primerextensiongel of fetal liver RNA from the HS I-V
`
`Human and mouse B-globin mRNAlevels were quantitated by
`solution hybridization with human B- and mouse B-globin-spe-
`cific oligonucleotides, as described (Towneset al. 1985). The
`values of percent expression per gene copy were calculated as-
`suming four mouse B-globin genes per cell. Mice used in this
`study (C57BL/6 x SJL} F2 have the Hbbé or single haplotype.
`The B-globin locus in this haplotype contains two adult p-
`globin genes (BS and §*) per haploid genome (Weaveret al. 1981).
`These mice also have two a-globin genes {al and «2) per ha-
`ploid genome (Whitneyet al. 1981; Erhart et al. 1987). Copies
`per cell of HS B transgenes were determined by densitometric
`scanning of the Southern blots illustrated in Fig. 3.
`«| ————_ x 100}.
`m B mRNA
`
`)
`(BERNA
`» ( RERRNAM Bene_oo x “*:100]
`
`m B mRNA/m f gene
`
`Page 335 of 547
`
`GENES & DEVELOPMENT
`
`317
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`

`
`
`Figure 5. Primer extension analysis of fetal liver RNA from
`HSI-V (30) 8 transgenic mice. As described in the legend to
`Fig. 4, 5 wg of fetal liver RNA was analyzed.
`
`gene were obtained, and all 13 animals expressed cor-
`rectly initiated human £-globin mRNAin fetal liver
`(Fig. 7}. Levels of expression ranged from 9.0 to 347% of
`endogenous mouse 8-globin mRNA. Whenthese values
`were corrected for transgene copy number, the average
`level of human f-globin expression was 49% of endoge-
`nous mouse £-globin expression (Table 1).
`The 13.0-kb Mlul—ClalI fragment containing HS I and
`HS II was then divided into a 5.8-kb Miul—BstEII frag-
`ment containing HS II and a 7.2-kb BstEII-Clal frag-
`ment containing HS I. Each of these fragments wasin-
`serted upstream of the human £-globin gene (Fig. 2} and
`injected into fertilized eggs. Unfortunately, no HS I B
`transgenic animals were obtained. However, nine an-
`imals containing the HS II (5.8) 8 construct wereidenti-
`fied by DNA dot hybridization, and seven of these nine
`animals contained intact copies of the transgene. Fetal
`liver RNA from all nine samples was analyzed by solu-
`tion hybridization and primer extension, and eight of
`nine animals expressed correctly initiated human p-
`globin mRNA(Fig. 8}. The single animal (5120) that did
`not express any human f-globin mRNA was the only
`
`
`
`Figure 6. Primer extension analysis of fetal liver RNA from
`HS I-V (22] B transgenic mice. As described in the legend to
`Fig. 4, 5 wg of fetal liver RNA was analyzed.
`
`318
`
`GENES & DEVELOPMENT
`
` Beeetae Soe
`
`Figure 7. Primer extension analysis of fetal liver RNA from
`HS L,I (13) B transgenic mice. As described in the legend to Fig.
`4, 5 wg of fetal liver RNA wasanalyzed.
`
`transgenic animals that did not express
`one of 51 HS
`the transgene. Thelevels of expression for samples 5140
`and 5153 were low but, as described above, both of these
`samples contained rearranged copies-of the transgene.
`Also, the fetal liver RNA of sample 5127 was somewhat
`degraded. The levels of human f-globin mRNA for
`samples 5127, 5118, 5132, 5131, 5148, and 5136 ranged
`from 8.0 to 108% of endogenous mouse B-globin mRNA.
`When these levels were corrected for transgene copy
`number, the values ranged from 6.0 to 84%, and the
`average level of human B-globin mRNAper gene copy
`was 40% of endogenous mouse B-globin mRNA(Table
`1).
`To begin to determine the minimal HSII sequencere-
`quired for high level expression, a 1.9-kb KpnI—Pvull
`
`
`
`Figure 8. Primer extension analysis of fetal liver RNA from
`HS Hf (5.8) B transgenic mice. As described in the legend to Fig.
`1, 5 pg of fetal liver RNA was analyzed. (Bottom) A 3-day expo-
`sure of the human f-globin, 98-bp primer extension productis
`shownin the insert. Samples 5140 and 5153 contained rear-
`ranged copies of the transgene {data not shown), and the RNA
`from sample 5127 was degraded slightly. Sample 5120 was the
`only one of 51 transgenic mice that contained an intact copy of
`the transgene but did not express any human B-globin mRNA.
`
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`

`

`Ue eg we eee wy ew
`
` oe nerobile
`
`ee)
`
`Figure 9. Primer extension analysis of fetal liver RNA from
`HSII (1.9) B transgenic mice. As described in the legend to Fig.
`4, 5 wg of fetal liver RNA was analyzed. Five micrograms of
`both fetal liver and brain RNA were analyzed for sample 5619.
`
`fragment containing HS I] wasinserted upstream of the
`human £-globin gene (Fig. 2) and tested for activity in
`transgenic mice. Four animals that contained intact
`copies of the transgene were obtained, andall four ex-
`pressed correctly initiated human B-globin mRNA in
`fetal liver (Fig. 9). The levels of human 6-globin mRNA
`ranged from 56 to 194% of endogenous mouse B-globin
`mRNA. Whenthese values were corrected for transgene
`copy number,
`the average level of human 6-globin
`mRNA was 40% of endogenous mouse f-globin mRNA
`(Table 1).
`Finally, the human @-globin gene without HSsites
`wasinjected into fertilized eggs and assayed for expres-
`sion in 16-day fetal liver. In this experiment, only 7 of 23
`mice that contained intact copies of the transgene ex-
`pressed human f-globin mRNA, and the levels of ex-
`pression ranged from 0.2 to 23% of endogenous mouse
`B-globin mRNA. Whenthese levels were corrected for
`transgene copy number, the average level of human p-
`globin mRNA was 0.3% of endogenous mouse f-globin
`mRNA(Table 1).
`
`B-Globin gene expression in transgenic mice
`
`Tissue specificity of HS B-globin transgene expression
`Fetal liver and brain RNA from the highest expressor of
`eachset of transgenic animals were analyzed for human
`B-, mouse a-, and mouse B-globin mRNAby primerex-
`tension to assess the tissue specificity of human p-
`globin gene expression. Data in Figure 10 and in the last
`two lanes of Figure 9 demonstrate that the human £-
`globin gene is expressed in fetal liver and not in brain.
`The small amount of human #-globin mRNAin the
`brain results from blood contamination because equiva-
`lent amounts of mouse a- and §-globin mRNAarealso
`observed in this nonerythroid tissue. Solution hybridiza-
`tion analysis demonstrated that the ratio of human £-
`globin mRNA to mouse B-globin mRNA wasvirtually
`identical in fetal liver and brain in all 50 HS B transgenic
`mice. These data strongly suggest that the HSsites act
`specifically in erythroid tissue to stimulate high levels
`of human £-globin gene expression in transgenic mice.
`Discussion
`
`Summary of HS B-globin expression
`
`A summary of the results presented aboveare listed in
`Table 1. In this study only 7 of 23 animals without HS
`sites expressed the transgene. In contrast, 50 of 51 an-
`imals that contained HSsites inserted upstream of the
`human f-globin gene expressed correctly initiated
`human £-globin mRNAinfetal liver and no expression
`was detected in fetal brain. These results, like those of
`Grosveld et al. (1987} with a construct containing HS
`I-VI B, suggest that the HS sites activate expression re-
`gardless of the site of transgene integration. However,
`expression is not
`totally position independent. The
`range of expression varied widely with all of the con-
`structs tested, and levels of human §-globin mRNA
`were not absolutely correlated with transgene copy
`number. Nevertheless, the average levels of expression
`per gene copy were high for all of the HS B-globin con-
`structs tested. The HS I—V (30) B and HS I-V (22) B con-
`structs were expressed at an average level of 108 and
`109%, respectively, of endogenous mouse B-globin per
`gene copy, andall other HS £ constructs were expressed
`
`
`
`Figure 10. Primer extension analysis offetal liver and
`brain RNAof HSBtransgenic mice. As described in the
`legend to Fig. 4, 5 wg of fetal liver and brain RNA from
`the highest expressor of each set of HS P transgenic
`mice were analyzed. The low level of human B-globin
`mRNAobserved in the brain is the result of blood con-
`tamination because equivalent levels of mouse a- and
`: B-globin mRNAsare also observedin this tissue.
`
`GENES & DEVELOPMENT
`
`319
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`

`Ryan et al.
`
`we eg ee eee ey eee
`
`at 40-49% of endogenous mouse @-globin per gene
`hance expression. Second, a very interesting deletion in
`copy. This high level of expression was obtained even
`a Hispanic ydf-thalassemic patient has recently been
`when a 1.9-kb fragment containing only HS II wasin-
`defined by C. Driscoll et al. (pers. comm.]. A 30-kb dele-
`serted upstream of
`the human 6-globin gene. The
`tion that ends 9.8 kb upstream of the e-globin genere- -
`average level of expression per gene copy for a human
`moves HS V—II but leaves HSIintact (Fig. 1). The pa-
`B-globin construct that did not contain HS sites was
`tient, who has a B° gene on this same chromosome,
`only 0.3% of endogenous mouse B-globin. This average
`makes no sickle hemoglobin. The datafrom this patient
`level of expression is 133-363 times lower than con-
`and the transgenic animal described above strongly sug-
`structs containing HSsites. Finally, we suspect that the
`gest that HS I cannot,byitself, stimulate expression of
`average level of expression for the HS I-VI B construct
`downstream globin genes.
`was lower than 100% per gene copy because only three
`animals were obtained.
`
`Role of individual HSsites
`
`Southern blots of fetal liver DNA from all 51 of the HS B
`transgenic mice generated in this study demonstrated
`head-to-tail tandem arrays of the transgene (data not
`shown}. Therefore, every animal contains at least one
`copy of the human #-globin gene that is flanked on ei-
`ther side by HSsites. This is true even for animals that
`contain one or fewer copies per cell of the transgene.
`These animals must be mosaics (Wilke et al. 1986] with
`multiple tandemly linked transgenes in only a fraction
`of their cells. Although the data demonstrate that HS VI
`is not required for high level expression, a copy of HS II
`or oneof the other upstream HSsites may substitute for
`HS VI wheninserted downstream ofthe B-globin gene in
`the tandem array. To determine whether a downstream
`HSsite is required for high level expression, animals
`containing a single copy of HS I—V B or HS I B will have
`to be produced.
`Wehavenotyet tested the activity of HS I, HS IV,or
`HS V,inserted individually upstream of the human p-
`globin gene. However, one or more of these sites may be
`active because transgenic animals that contain HS I-V
`consistently express higher levels of human ®-globin
`mRNAthan animals that contain HS I and HS Hl or HS II
`alone. Individual sites and various combinationsof sites
`are now being tested to determine the minimal se-
`quences required for maximal expression. As individual
`sites may be functionally redundant,it will also be inter-
`esting to test constructs containing multiple copies of
`HS II inserted upstream of the human B-globin gene to
`determine whether multimers of an individual site can
`substitute for HS I-V.
`Because HS I B transgenic animals were not obtained,
`we do not know whether HSI alone can stimulate p-
`globin gene expression. However,
`two pieces of data
`argue strongly that HS I is not sufficient to enhance ex-
`pression. First, we have demonstrated recently that the
`human a-globin gene is expressed at high levels in trans-
`genic mice when placed downstream of HS I and HSII
`(Ryan et al. 1989). Of 12 HSI, HSH, a-globin mice, 11
`expressed correctly initiated human a-globin mRNA
`specifically in erythroid tissue, and the average percent
`expression per gene copy was 57% of endogenous mouse
`6-globin mRNA.Thesingle animal that did not express
`human a-globin mRNA had intact copies of HS I a-
`globin, but the HS Il site had been deleted uponintegra-
`tion. This result suggests that HS I alone cannot en-
`
`320
`
`GENES & DEVELOPMENT
`
`H§site effect on other genes
`
`The effects of erythroid-specific HS sites on other tissue
`specifically expressed genes has not been tested. How-
`ever, the experiments of Nandi et al. (1988) strongly sug-
`gest that the SV40 promoter can be dramatically in-
`fluenced by HS sites. Murine erythroleukemia (MEL)
`cells containing human chromosome 11 were trans-
`fected with a construct containing a modified human B-
`globin gene and an SVneo gene. G418-resistant cells
`were identified that contained this construct inserted
`specifically into the human £-globin locus or at nonspe-
`cific chromosomalsites. When these cells were induced
`to differentiate with dimethylsulfoxide (DMSO}, SVneo
`mRNAwasinduced to high levels in cells with site-spe-
`cific integrants but not in cells with random integrants.
`These results strongly suggest that expression from het-
`erologous promoters can be greatly enhanced by the HS
`sites. We have also demonstrated that SVneo expression
`is induced to high levels in MEL cells transfected with
`cosmids containing HS I-V £ linked to the SVneo gene
`{unpubl.).
`
`Human §-globin domain
`
`Several groups have suggested that HS sites mark the
`boundaries of the human @-globin domain andthat these
`sites are responsible for opening the B-globin domain
`specifically in erythroid tissue (Tuan et al. 1985; For-
`rester et al. 1986, 1987; Grosveldet al. 1987}. Forrester et
`al. (1987) have demonstrated recently that these HSsites
`are formed in human fibroblasts that have been fused
`with MELcells. These hybrids synthesize high levels of
`human -globin mRNA. Presumably,
`trans-acting
`factors present in MELcells interact with the hypersen-
`sitive site sequences both upstream and downstream of
`the human f-globin locus and organize the previously
`closed chromatin domain into an open domain. There-
`fore, Forrester et al. (1987) have suggested that the se-
`quences be called ‘locus activating regions,’ or LARs.
`Similarly,
`in the developing human embryo,
`trans-
`acting factors present in early erythroid cells may in-
`teract with hypersensitive site sequences and activate
`the B-globin locus for expression.
`
`Modelfor developmental regulation
`
`Choi and Engel (1988) have demonstrated recently that
`sequences at the immediate 5’ end of the chicken 8£-
`
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`

`ee ey re ee my ee
`
`B-Globin gene expression in transgenic mice
`
`al. 1985) were kindly provided by Oliver Smithies, and a \ clone
`containing HS VI (A4] was kindly provided by Don Fleenor and
`Russell Kaufman. A 1.9-kb HindIII fragment containing HS II
`was prepared from 5’eIII and subcloned into pUC19. A 1.3-kb
`BamHI-HindIll fragment from this plasmid was then used to
`screen a human placenta genomic library in EMBL 4 (Strata-
`gene} and several clones that overlapped with 5’elll were iso-
`lated. One clone that contained a 17.5-kb insert extended ~11.0
`kb upstream of the EcoRI site at the 5’ end of the 5’elll clone.
`This new clone, which was designated 5’e[V, contained HS V.
`Cosmid clone HS I~V (30) 8 was constructed as follows. A
`17-kb Sall—Mlul fragment was prepared from 5‘eIV; the Sall site
`was from the EMBL 4 Sall—BamHIcloning site, and the Mlul
`site was a natural site in the insert. This 17-kb fragment con-
`tained HS V, HS IV, and HS Ill. A 13-kb Miul—Clal fragment
`containing HSII and HS I wasprepared from 5’ell. These two
`fragments were inserted into the cosmid vector pCV001 (Lau
`and Kan 1983} in a four-way ligation. Theleft arm was a 9.0-kb
`Mlul--Sall fragment obtained from pCVO001; the Mlul site was
`destroyed by $1 digestion. This fragment contained a cossite,
`an ampicillin-resistance gene, a ColEI origin, and the SVneo
`gene. The right arm was a 6.6-kb Clal—Hindlil fragment that
`contained the human -globin gene on a 4.1-kb Hpal-Xbal
`fragment and a cos site from pCVO01 on a 2.5-kb Sall—HindII
`fragment. The Hpal and Xbalsites oneither side of the B-globin
`gene were changed to Clal and Sall, respectively, in the right
`arm plasmid.
`
`
`
`globin gene are involved in temporal specificity in tran-
`sient expression assays. These sequences apparently
`bind factors that influencethe ability of this promoter to
`compete with the e-globin gene promoter for interac-
`tions with a single erythroid enhancer (Choi and Engel
`1988; Nickol and Felsenfeld 1988] located in the chicken
`B-globin locus. Although similar mechanisms may be
`involved in developmental stage-specific expression of
`human globin genes,
`the situation is probably more
`complex. The major determinants of erythroid tissue
`specificity in humans appear to be the HS sequences.In
`fact, these sequencescarry out two importantfunctions:
`They organize the entire 8-globin locus for expression
`specifically in erythroid tissue, and they act as an en-
`hancer to direct high level expression. These two sepa-
`rate but related functions are evident in the experiments
`described above. First, the HS sites increase the fraction
`of transgenic animals that express the human £-globin
`gene. Of 51 HS §-globin mice, 50 expressed the trans-
`gene specifically in erythroid tissue compared with 7 of
`23 animals containing the ®-globin gene alone. Appar-
`ently, the HS sequences ensure that the transgene will
`be in an open chromatin domain regardlessof the site of
`integration. Second, HS sites stimulated the average
`level of B-globin gene expression 133- to 363-fold com-
`pared to the average level of the B-globin gene alone.
`HSV OHSIV HSI
`MSIE HSI
`Therefore,
`these: sequences constitute a powerful en-
`$$h $$
`hancer that may work in concert with enhancers ‘in and
`>
`>
`surrounding individual genes.
`cas
`Cla
`Miu t
`ori Salt
`COS
`Although human @-globin genes in transgenic mice
`HHO +f
`1
`13 kb
`L
`HHH 17 kb
`
`Amp=$Vnco Bglix Kpal Sal 1
`
`
`are expressed specifically in adult erythroid tissue
`without HSsites, high levels of correctly regulated ex-
`pression may require interactions between HS se-
`quences, promoters, and proximal enhancers. A model
`for globin gene regulation can be envisioned that incor-
`porates the two important functions of HS sites and the
`concept of competition between various regulatory se-
`quences. HS sequences could be activated in early ery-
`throid cell precursors and organize the entire B-globin
`locus into an open chromatin domain that is stable
`throughout development. Within the open domain,pro-
`moters and enhancers in and surrounding thee-, y-, and
`B-globin genes could then compete forinteractions with
`the HS master enhancer to determine which of these
`genes will be expressed. Promoter and proximal en-
`hancer binding factors synthesized in yolk sac, fetal
`liver, and bone marrow could influence these competi-
`tive interactions either positively or negatively and sub-
`sequently determine developmental specificity. Trans-
`genic mouse experiments with constructs containing
`human e-, y-, and B-globin genes inserted separately or in
`various combinations downstream of
`the HS sites
`should help define important interactions between regu-
`latory sequences and should, in general, provide mean-
`ingful insights into the complex mechanismsthat regu-
`late multigene families during development.
`Methods
`
`These four fragments were ligated in a 2: 1 : 2 vector arms to
`inserts and packaged (Gigapack Gold; Stratagene). Escherichia
`coli ED8767 was then infected with the packaged cosmids and
`plated on ampicillin plates. Large-scale cultures of ampicillin-
`resistant colonies were grown and cosmids were prepared by
`standard procedures (Maniatis et al. 1982).
`The HS I-V (22) B cosmid was constructed as follows. A
`12-kb Bgill fragment containing HS V, HS IV, HS If, and HS fi
`was subcloned from HS I-V {30} B into a modified pUC
`plasmid, and a 10.7-kb Sall—Kpnl fragment containing HS V,
`HSIV, and HS IU wasprepared from this plasmid. The Sail site
`of this fragment was from the pUCpolylinker, and the Kpnl
`site was a natural site in the insert. A 10.9-kb KpnI—Clal frag-
`mentcontaining HS II and HSI was isolated from 5’el and sub-
`cloned into a modified pUC plasmid. The 10.7-kb Sall—Kpnl
`fragment containing HS V, HSIV, and HSIl wasligated to the
`10.9-kb KpnI-Clal fragmentcontaining HS II and HSI and the
`two cosmid vector arms described above. The ligation mixture
`was packaged, ED8767 cultures were infected,’ and cosmids
`were prepared from ampicillin-resistant colonies.
`HS I-VI 8 wasprepared as follows. A 12.0-kb Hpal-BamHI
`fragment containing HS VI was subcloned from \4into a modi-
`fied pUC19 plasmid and then isolated from this plasmid as a
`12.0-kb Xhol—Sall fragment. This fragment wascloned into the
`Sall site downstream of the human f-globin gene in the right-
`arm plasmid described above. The right-arm plasmid was then
`linearized with Clal and dephosphorylated with calf intestinal
`phosphatase (Boehringer~Mannheim}. This 21-kb right-arm
`fragment and the 9.0-kb Mlul—Sall left-arm fragment described
`above were ligated with the 10.7-kb Sail—Kpnl fragment con-
`taining HS V, HS IV,and HSIII and the 10.9-kb KpnI—Clal frag-
`
`GENES & DEVELOPMENT
`
`321
`
`Construction of HS f-globin clones
`
`Lambda clones containing HSsites I-IV (5’ell and 5’elll; Li et
`
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`ceee
`
`Ryanet al.
`
`ment containing HS II and HS J in a 2: 1:1 molar ratio of
`vector arms to inserts. The ligation mixture was packaged,
`ED8767 cultures were infected, and cosmids were prepared
`from ampicillin-resistant colonies.
`HSLT (13) B was derived from HS I-V (22) p after digestion
`with Mlul and Sail. HS II (5.8) B and HS II (1.9) 6 were con-
`structed by subcloning the 5.8-kb Mlul—BstEIl fragmentor the
`1.9-kb Kpnl—Pvull fragment into modified pUC plasmids con-
`taining the human f-globin gene.
`
`Sample preparation and microinjection
`All of the constructs were removed from vector sequences by
`digestion with the appropriate enzymes and isolated on low-
`gelling temperature agarose (FMC)gels. Gel slices were melted,
`extracted twice with phenol [buffered with 0.1 M Tris-HCl (pH
`8.0), 1.0 mm EDTA], once with phenol/chloroform, and once
`with chloroform andprecipitated with ethanol. After resuspen-
`sion in TE [10 mm Tris-HCl(pH 8.0], 1.0 mm EDTA|, thefrag-
`ments were again extracted with phenol, phenol/chloroform,
`and chloroform and precipitated with ethanol. The purified
`fragments were washed with 70% ethanol, resuspended in
`sterile TE, and microinjected into the male pronuclei of F2 hy-
`brid eggs from C57BL/6 x SJL parents as described by Brinster
`et al. (1985).
`
`DNA analysis
`
`Total nucleic acids were prepared from 16-day fetal liver and
`brain, as described previously {Brinster et al. 1985). Samples
`that contained the injected constructs were determined by
`DNAdot hybridization of brain nucleic acids with human B-
`globin and HSIl-specific probes that were labeled by extension
`of random primers {Feinberg and Vogelstein 1983). The human
`B-globin probe was a 790-bp Hinfl fragment from IVS2, and the
`HS Il probe was a 1.9-kb HindIII fragment spanning the HS II
`site. Hybridizations were performed at 68°C for 16 hr in 5x
`SSC, 5x Denhardt's solution, 100 ug/ml herring sperm DNA,
`and 0.1% SDS. Filters were washed three times for 20 min each
`at 68°C in 2x SSC, and 0.1% SDS and for 20 min at 68°C in
`-0.2x SSC and 0.1% SDSif necessary to reduce background.
`For Southern blots, 10 wg of fetal liver DNA from animals
`that were positive with HS II and/or B-globin probes were di-
`gested with BamHI and Pstl, electrophoresed on 1.0% agarose
`gels, blotted onto nitrocellulose, and hybridized with the B and
`HSII probes described above. The hybridization conditions for
`Southem blots were the same as described for DNA dots.
`
`RNA analysis
`
`RNA wasprepared from total nucleic acids by digesting the
`sample with DNase I (Worthington, RNase-free] at 10 wg/mlfor
`20 min at 37°C in 10 mM Tris-HCl (pH 7.5), 10 mm MgCl, and
`50 mM NaCl. The reaction was stopped with EDTA, and the
`sample was digested with proteinase K (100 g/ml} for 15 min
`at 37°C. After digestion, RNA was purified by phenol/chloro-
`form and chloroform extraction, precipitated with ethanol, and
`resuspendedin TE.
`Quantitation of human and mouse £-globin mRNA wasde-
`terminedby solution hybridization with oligonucleotide probes
`as described (Townes et al. 1985b}. Primer extensions were per-
`formedas described by Townesetal. (1985a,b}, except that only
`5 wg offetal liver or brain RNAs were analyzed andthree oli-
`gonucleotides were used in each reaction. The human 8 primer
`5'-AGACGGCAATGACGGGACACC-3' corresponds to se-
`quences from +78 to +98 of the human B-globin gene. The
`
`322
`
`GENES & DEVELOPMENT
`
`mouse a primer 5’-CAGGCAGCCTTGATGTTGCTT-3’corre-
`sponds to sequences from +45 to +65 of the mouse al- and
`a2-globin genes, which are identical in this region. The mouse
`B primer 5’-TGATGTCTGTTTCTGGGGTTGTG-3’ corre-
`sponds to sequences +31 to +53 of the mouse B*-globin gene.
`Although there are 2-bp differences in the BS and Bt genesin the
`region covered by this oligonuceotide, comparison of solution
`hybridization results {obtained with a different oligonucleotide
`that is perfectly complimentary to BS and Bt; see Townes et al.
`1985b) with primer extension data suggests that the primer an-
`neals with equal efficiency to B5- and Bt-globin mRNA under
`the hybridization conditions used.
`
`Acknowledgments
`We thank Oliver Smithies for \ clones 5’elI and 5'elll and Don
`Flenor and Russell Kaufman for 44. We especially thank Cathy
`Driscoll for communicating results on the Hispanic thalas-
`semia prior to publication. We also thank Josef Prchal for pro-
`viding human reticulocyte RNA and Jeff Engler for synthe-
`sizing the human £- and mousef-globin oligonucleotides. This
`work was supported, in part, by grants HL-35559, HD-09172,
`and HD-17321 from the National Institutes of Health. T.M.R.
`is a predoctoral trainee supported by National Institutes of
`Health grant T32 CA-09467,
`
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