PRODUCTION OF HUMAN HEMOGLOBIN IN TRANSGENIC PIGS

Abstract: The present invention relates to the use of transgenic pigs for the production of human hemoglobin in which, in certain embodiments, the pig beta globin promoter is used to facilitate the expression of human hemoglobin. The transgenic pigs of the invention may be used as an efficient and economical source of cell-free human hemoglobin that may be used for transfusions and other medical applications in humans.

Description: PRODUCTION OF HUMAN HEMOGLOBIN IN TRANSGENIC PIGS * 5 1. INTRODUCTION The present invention relates to the use of transgenic pigs -for the production of human hemoglobin. -The transgenic pigs of the invention may be used as an e ficient and economical source of cell- 20 free human- hemoglobin that may be used for transfusions and other medical applications in humans. 2.ι BACKGROUND OF THE INVENTION ..-• 2.1. HEMOGLOBIN 15 ■ _ . < , < > Oxygen absorbed through the lungs is carried by hemoglobin inured blood cells for delivery to tissues throughout 'the body. At high oxygen tensions, such as those- found in >the proximity of the lungs, oxygen, binds to hemoglobin, but is released in areas 20 of J°w oxygen tension, where it is needed. Each .hemoglobin molecule consists of two alpha globin and "two beta globin subunits. Each subunit, Jn turn., is noncovalently associated with an iron-containing.>heme group capable of carrying an 25 oxygen molecule. Thus, each hemoglobin tetramer is capable of binding four molecules of oxygen. The subunits work together in switching between two conformational states to facilitate uptake and release of oxygen at the lungs and tissues, respectively. 30 This effect is commonly referred to as heme-he e interaction or cooperativity. The hemoglobins of many animals are able to interact with biologic effector molecules that can further enhance oxygen binding and release. This 35 enhancement is manifested in changes which affect the allosteric equilibrium between the two conformational states of hemoglobin. For example, human and pig hemoglobin can bind 2, 3 diphosphoglycerate (2,3 DPG) , which influences the equilibrium between the two conformational states of the tetramer and has the net, effect o£ powering the overall affinity for oxygen at 5. the, tissue level. , As a result, 2,3-DPG increases the efficiency. of oxygen delivery to the tissues. t _ 2.2. GLOBIN GENE EXPRESSION ., - , , Hemoglobin protein is expressed in a tissue 0 s P^?ifip manner in red blood cells where it accounts for sgproximately ninety percent of total cellular protean. Thus, red blood cells, which have lost their nucJeus and all but a minimal number of organelles, arp effectively membrane-enclosed packets of 5_ heppgjqbin ι dedicated to oxygen transfer. „ , , ι , Humans and various other species produce^ different types of hemoglobin during embryonic, fetal, and adult developmental periods. Therefore, the factory that influence globin gene expression must be 0 able to achieve tissue specific control, quantitative control, and develop entally regulated control of „ , globin expression. • , ,,. Human globin genes are found in_clusters on chromosome 16 for alpha (α) globin and chromosome 11 fqr beta (β) globin. The human beta globin gene cluster consists of about 50 kb of DNA that includes ope embryonic gene encoding epsilon (e) globin, two fetal genes encoding gamma (7) G and gamma A globin, an two adult genes encoding delta (<5) and beta (j3) globin, in that order (Fritsch et al., 1980, Cell _ 19:959-972). It has been found that DNA sequences both upstream and downstream of the β globin translation initiation site are involved in the regulation of β globin gene expression (Wright et al., 1984, Cell j3£:263) . In particular, a series of four Dnase I super hypersensitive sites (now referred to as the locus control region, or LCR) located about 50 kilobases upstream of the human beta globin gene are extremely important in eliciting properly regulated beta globin-loόu≤ expression (Tuan et al. , 1985, Proc. Natl. Acad. Sci. U.S.A. 82:1359-1363; PCT Patent Application ' WO a901517 by Grosveld; Behringer et al., 1989, Science 245:971-973; Enver et al., 1989, Proc. Natl. Acad. Sci. U.S.A. .8^:7033-7037; Hanscombe et al., 1989, Genes Dev. 3 . :1572-1581; Van Assendelft et al., 1989, Cell J6:967-977; Grosveld et al., 1987, Cell 51:975-985) . ' 2.'3.. THE NEED FOR A BLOOD SUBSTITUTE Recently, the molecular aspects of globin gene expression have met with even greater interest as researchers iiave attempted to use genetic engineering to produce a synthetic blood that would avoid the < pitfalls -of donor generated blood. In 1988, between 12 million and 14 million units of blood were used in the United States alone (Andrews, February 18, 1990, New York'Times , an enormous volume precariously dependent on volunteer blood donations. About 5 percentOf donated blood is infected by hepatitis virus (Id-) and, 'although screening procedures for HI-V infection are generally effective, the prospect of contracting transfusion related A.I.D.S. remains a much feared possibility. Furthermore, transfused blood must be compatible with the blood type of the transfusion recipient; the donated blood supply may be unable to provide transfusions to individuals with rare blood types. In contrast, hemoglobin produced by genetic engineering would not require blood type matching, would be virus-free, and would be available in potentially unlimited amounts. Several research groups have explored the possibility of expressing hemoglobin in microorganisms. For example, see International Application No. PCT/US88/01534 by Hoffman and Nagai, which presents, in working examples, production of human globin protein- in E. coli. - ' ' 5 . , , 2.4. TRANSGENIC ANIMALS A transgenic animal is a non-human animal containing at least one foreign gene, called a transgene, in its genetic material. Preferably,' the traπsgene is contained in the animal's germ line such 0 , that it can be transmitted to the animal's offspring. A number of techniques may be-used to introduce the 11 transgene into an animal's genetic material) -' including, but not limited to, microinjection of the transgene into pronuclei of fertilized eggs and ' _3- manipulation of embryonic stem cells (U.S. 'Patent-No 1 . 4;873,191 by Wagner and Hoppe; Palmiter and Brinster, 1986, Ann. Rev. Genet. ^0:465-499; French Patent -' -< ■ ' Application 2593827 published August 7, 1987). ■ Transgenic animals may carry the transgene in all "-"- 0 their cells or may be genetically mosaic. Although the majority of studies have involved transgenic mice, other species of transgenic animal have also been produced, such as rabbits, )( " ' ' sheep-, pigs (Hammer et al., 1985, Nature 315:680-683) 5 and chickens (Salter et al., 1987, Virology 157:236- 240) . Transgenic animals are currently being developed to serve as bioreactors for the'production of useful pharmaceutical compounds (Van Brunt; 1988, Bio/Technology 1:1149-1154; Wilmut et al. , 1988, New' 0' Scientist (July 7 issue) pp. 56-59). ι ' Methods of expressing recombinant protein via transgenic livestock have an important theoretical 1 advantage over protein production in recombinant bacteria and yeast; namely, the ability to produce 5 large, complex proteins in which post-translational modifications, including glycosylation, phosphorylation, subunit assembly, etc. are critical for the activity of the molecule. in practice, however, " the creation of transgenic livestock has proved " problematic. Not only is it technically difficult to produce transgenic 5 embryos, but mature transgenic ariimals that produce significant quantities of recombinant protein may prove inviable. In pigs in particular, the experience has been that pigs carrying a growth hormdhe encoding transgene (the only transgene introduced into pigs 0 prior to the present invention) suffered from a number of health problems, including severe arthritis, lack of coordination in their rear legs, susceptibility to stress, anόestrus in gilts ari lack of libido in boars (Wilmut et al., supra) . This is in ontrast to 5 transgenic mice carrying a growth hormόne J transgene, which appeared to be healthy (Palmiter et aϊJ,' 1982, Nature 300:611-615). Thus, prior to the''present - " " invention, healthy transgenic pigs (which efrϊbiently express their transgene(s) ) Had not been produdέcL. b ' ' 2.5. EXPRESSION pfr G£OBIN GENES IN TRANSGENIC ANIMALS Transgenic mice carrying human gϊόbin transgenes have been used in studying the' molecular biology of globin gene expression. A hybrid '" ' 5 mouse/human adult beta globiri gene was described by Magram et al. in 1985 (Nature ' 315:3 8-34O) . Kollias et al. then reported regulated expression of human gamma-A, beta, and hybrid beta/gamma globin genes in transgenic mice ' (1986, ' Cell 6:89-94)'. ; Tr nsgehib 0 mice expressing human fetal gamma gldfeih were studied by Enver et al. (1989, Proc. NatL ' cad. Sci. U.S.A. 6:7033-7037) and Cohstantbulakis et al. (1991, Blood 22:1326-1333). Autonomous developmental control of human embryonic globin gene switching in transgenic 5 mice was observed by Raich et aϊ. ' (1990, Science 250:1147-1149) . Transgenic mouse models for a variety of disorders of hemoglobin or hemoglobin expression have been developed, including sickle cell disease (Rubin et al-. , 1988, Am. J. Human Genet. 42:585-591; Greaves et al., 1990, Nature 343:183^185; Ryan et al., 1990, 5 Science 247:566-568; Rubin et al., 1991, J. Clin. Invest. . 82:639-647); thalassemia (Anderson et al., 1985, Ann. New York Acad. Sci-. (USA) 445:445-451; Sorenson et al., 1990, Bloo '25:1333-1336) ; and hereditary persistence of fetal hemoglobin (Tanaka et 0 al., 1990, Ann. New York-Acad. Sci. (USA) 612:167- 178) . • ' Concurrent expression of human alpha and beta globin has 1 led to the production of human hemoglobin in transgenic mice (Behringer et al. , 1989, 5 Science 245 971-973 ; Townes et al., 1989, Prog. Clin. Biol. Res. 316A:47-61; Hanscombe et al., 1989, Genes Dev. 3 . :1572-1581) . It was observed by Hanscombe et al. (supra) that transgenic fetuses with high copy numbers of a transgene encoding alpha but not beta o globin exhibited severe- anemia and died prior to birth. Using a construct with both human alpha and beta globin genes under the control of the beta globin LCR, live mice' with low copy numbers were obtained (Id.). Metabolic labeling! experiments showed balanced mouse globin synthesis, but imbalanced human globin synthesis, with- an alpha/beta biosynthetic ratio of about 0.6 (Id. ) . 3. SUMMARY OF THE INVENTION The present invention relates to the use of transgenic pigs for the production of human hemoglobin and/or human globin. It is based, at least in part, on the discovery that transgenic pigs may be generated that express human hemoglobin in their erythrocytes and are healthy, suffering no deleterious effects as a result of heterologous hemoglobin production. In particular embodiments, the present invention provides for transgenic pigs that express human globin genes. Such animals may be used as a. particularly efficient and economical source of <human hemoglobin, in light of (i) the relatively short periods of gestation and sexual maturation in pigs; (άi) the size and frequency of litters, (iii) the relatively large size of the pig which provides proportionately large yields of hemoglobin; and (iv) functional similarities between pig and human hemoglobins in the regulation of oxygen binding affinity which enables the transgenic pigs to remaih healthy in the presence of high levels of human hemoglobin. The present invention also provides for recombinant nucleic acid constructs that may be used to generate transgenic pigs. In preferred ι embodiments, such constructs place the human-alpha and beta globin genes under the same promoter so as to avoid deleterious effects of globin chain imbalance and/or titration of transcription factors due to* <- constitutive j8-globin promoter activity in an inappropriate cell type (e.g. a primitive' < > erythrocyte)- In other preferred embodiments of the invention, the constructs comprise the pig adult.beta globin gene regulatory region, comprising the promoter or the 3' region of the pig beta globin ' gene. In an additional embodiment, the present invention provides for a hybrid hemoglobin that comprises human α globin and pig β globin. The , hole blood from transgenic pigs expressing this hybrid hemoglobin appears to exhibit a P, 0 that is advantageously higher than that of native human or pig blood. The present invention also provides for a method of producing human hemoglobin comprising (i) introducing a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the genetic material of a pig so as to create a transgenic pig that expresses human hemoglobin in at least some of its red blood cells; (ii) collecting red blood cells from the transgenic pig; (iii) releasing the contents of the collected red blood cells; and (iv) subjecting the released contents of the red blood cells to a purification prpcedure that substantially separates human hemoglobin from pig hemoglobin. In a preferred embodiment of the invention, human hemoglobin may be separated from pig hemoglobin by DEAE anion exchange column chromatography. . „ 4. DESCRIPTION OF THE FIGURES Figure l. Recombinant nucleic acid constructs. A. Construct aaβ (the "116 construct); B. Construct pβ (.the "185" construct) ; C. Construct βp (the "290" construct) ; D. Construct epfjSα; E. Construct frpeαpβ; F. Construct αp3 carrying a j8l08 Asn -> Asp mutation (the "hemoglobin Yoshizuka construct") ; G. Construct αp/3 carrying a 0108 Asn -> Lys mutation (the "hemoglobin Presbyterian construct"); H- Construct αp3(Δα) coinjected with LCR α (the "285" construct) ; I. Construct αp/3 carrying an αl34 Thr -> Cys mutation (the "227" construct) ; J. Construct apβ carrying an αl04 Cyε-> Ser mutation (the "227" construct) , a (893 Cys -> Ala mutation, and a βll2 Cys -> Val mutation (the "228" construct) ; K. Construct αpS (the "263" construct) ; and L. Construct αp<5(Δα) coinjected with ;LCR α (the "274" construct) ; M. Construct LCR a coinjected with LCR eβ (the "240" construct); N. Construct αpβ carrying a β.61 Lys -> Met mutation (the "Hemoglobin Bologna" construct) ; O. Construct LCR eaβ (the "318" construct) ; P. Construct LCR aeβ (the "319" construct) ; Q. Construct LCR α e/3 (the "329" construct) ; R. Construct LCR ae ( p *βp) β (the "339" construct) ; S» Cpnstruct. txpβ carrying an 75 Asp -> Cys mutation- (the "340" construct); T. Construct αpβ carrying an α4<2 Tyr -> Arg mutation 5 (the "341" construct); U. Construct LCR eβ (the "343" construct) ; V. Construct LCR eβ (the "347" construct); W. Construc , αp/? carrying an α42 Tyr -> Lys mutation; X. Construct pβ carrying an 42 Tyr -> Arg mutation; and aι 399 Asp -> Glu 0 mutation; Y. Construct pβ carrying an α42 Tyr -> Lys mutation; and,_a S99 Asp -> Glu mutation. Figure 2. Transgenic pig. Figure 3. _ Demonstration of human hemoglobin expression in transgenic pigs. A. Isoelectric 5 focusing gel analysis. ,B . .. Triton-acid urea gel of iemolysates o ^ d blood :ells representing human blood (lane !); bloo.d, £rom transgenic pig . 12-1 (lane 2) ,, '9- 3, (lane 3>, and 6-3 (lane 4) ; and pig blood (Jane,5). shows under-expression of o human β globin relative <to .human ,α globin in the transgenic animals. Figure 4. Reparation of human hemoglobin and pig hemoglobin by DEAE ,chromatography. A. Hemolyzed mixture of human and. pig red bloo cells; B. 5 Hemolysate ^ of ,red blood cells collected from transgenic pig 6-3. _,,C. Human and mouse hemoglobin -do n°ft separate ^ by DEAE chromatography under these conditions, D. Isoelectric focusing of human hemoglobin r purified from pig hemoglobin. 0 Figure 5. Isoelectric focussing gel of reassociated pig hemoglobin (lane 1) ; reassociated pig/human hemoglobin mixture (lanes .2 and 4) ; reassociated human hemoglobin (lane 3) ; and transgenic pig hemoglobin (lane 5) . 5 Figure 6. Separation of human hemoglobin by QCPI chromatography. Figure 7. Oxygen affinity of transgenic hemoglobin. Figure 8. DNA -sequence of the pig adult beta globin gene regulatory region, including the promoter region. Sequence extending tp 869 base pairs upstream of the ATG initiator codon (boxed) of the pig beta globin gene is shown., The position of the initiation of mRNA, the .cap site, is indicated by an arrow. The sequences corresponding to GATA transcription factor binding sites are underlined. Figure 9,. Comparison of pig (top) and human (bottom) beta globin regulatory sequences. Differences in the two sequences are marked by asterisks. Figure 10. , Qraph depicting the percent homology between pig and human adult beta globin gene , , regulatρt;y sequences, with base pair distance from ,the initiator codon mapped on the abscissa. A comparison dθf mouse and human sequences is also shown (dotted line with error bar) . Figure 11,.. iføp of plasmid pgem5/Pig/3Pr(k) which contains the DNA sequence depicted in Figure 8. Figure 12. Representation of the 339 and 354 cassettes or the production of human hemoglobin in transg,enic pigs. Figure 13.- Map of plasmid pSaf/Pige (k) , containing _ ,, the pig e gene. Figure 14. .Representation of the 426 and 427 expression cassettes for the production of e pιg β hum ™ and g buπ n hemoglobins in transgenic pigs. Figure 15. Iso-electric focussing gel of hemoglobin produced by .transgenic pig 70-3, which carries the 339 construct, and by transgenic pig 6-3, which carries, the 116 construct. Human hemoglobin is run as a standard. Figure 16. Map of plasmid pig3'/3 containing the 3 ' end of the pig beta globin gene. Figure 17. Transgenic pigs obtained from construct "339" (See Figure 1R) . Levels of human hemoglobin expression and copy number are shown. Figure 18. Isolelectric- focussing gel of hemoglobin levels in transgenic pigs obtained 'using construct "339". Figure 19. Isoelectric focussing gel demonstrating levels of hemoglobin expression in representative transgene positive 38-4 offspring carrying the "185" construct (or apβ construct-; see Figure IB). Figure 20. Molecular modeling of hybrid human α/pig β and human α/human β hemoglobin molecules. β subunits are in blue, α subunits in red. Above the middle helix of the β human (blue) one can see a gap in the green contour (see arrow) . In the hybrid this gap is filed in. This difference is due to a change at j8ll2 Cys—->Val>where Valine contributes to greater hydrophobic interactions. Figure 21. Molecular modeling demonstrating the differences at the α,jS, interface between a β globin containing Cys at position 112 (the yellow molecule) and a β globin ith Val at position 112 (the white molecule) . Cys< is yellow, Val is white and the opposing interface is red. Val is flexible. One- arm of its branch can easily - move for a nearly perfect fit against the α subunit residues. The yellow Cys is slightly further allowing for a small gap (see arrow) . Biosyn's standard default Van der Waal's distance was used. Figure 22. Purification of Hb Presbyterian from transgenic pig hemosylate. Figure 23. Characterization of purified Hb Presbyterian by HPLC showing separation of the heme moiety, pig α globin ("p alpha") , human beta globin ("h beta") , human alpha globin ("h alpha") and pig beta globin ("p beta") . Figure 24. Oxygen binding curve for Hb Presbyterian. Figure 25. Purification of Hb Yoshizuka from transgenic pig hemolysate. 5. DETAILED DESCRIPTION OF THE INVENTION The present -indention provides for a method of producing human ' hemoglobin that utilizes transgenic pigs, novel globin~encoc!ling nucleic acid constructs, and transgenic pigs"that express human hemoglobin. For purposes of clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections: (i) preparation of globin gene constructs; (ii) preparation of transgenic pigs; ' ϋi) preparation of human hemoglobin and its separation from pig hemoglobin; and (iv) f ' preparation of human/pig hybrid <■- ' hemoglobin. ■ 5.1. PREPARATION OF GLOBIN GENE CONSTRUCTS The present invention provides for a method of producing human globin and/or hemoglobin in transgenic pigs. Human hemoglobin is defined herein to refer to hemoglobin formed by globin chains encoded by human globin genes (including alpha, beta, delta, gamma, epsilon and zeta genes) or- variants thereof which are naturally occurring or the products of genetic engineering-!. Such variants are at least about ninety percent homologous in amino acid sequence to a naturally occurring human hemoglobin. In preferred embodiments, the human hemoglobin of the invention comprises a human alpha globin and a human beta globin chain. The human hemoglobin of the invention comprises at least two different globin chains, but may comprise more than two chains, to form, for example, a tetrameric molecule, octa eric molecule, etc. In preferred embodiments of the invention, human hemoglobin consists of two human alpha globin chains and two human beta globin chains. As discussed' infra, the present invention also provides for hybrid hemoglobins comprising human α globin and pig β globin. > ■ According to particular embodiments of the present invention, at least one human globin gene, such as.a'hu an lalpha and/or a human beta globin gene, under the cdntrαl of a suitable promoter or promoters, is inserted!into i he genetic material of a pig so as to create a transgenic pig that carries human globin in at least some of its red blood cells. This requires the preparation of appropriate recombinant nucleic acid sequences. In preferred embodiments of the invention, both human a and human β genes are expressed. In an alternative embodiment, only human α globin or human β globin is expressed. In further embodiments,^ -human embryonic or fetal globin genes are expressed iorJ re used as developmental expression regulators of > adult genes. "Human alpha and beta globin genes may be obtainedifrom publicly available clones, e.g. as described in Swanson et al., 1992, Bio/Technol. 10:557-559. Nucleic acid sequences encoding human alpha and beta globin proteins may be introduced into an animal via two different species of recombinant constructs, one which encodes human alpha globin, the- other encoding human beta globin; alternatively, and preferably, both alpha and beta-encoding sequences may be comprised in the same recombinant construct. The pig epsilon globin gene is contained in plasmid psaf/pig e (k) (Figure 13), deposited with the ATCC and assigned accession number 75373. t i A suitable promoter, according to the invention, is a promoter which can direct transcription of human alpha and/or beta globin genes in red blood cells. Such a promoter is preferably •Selectively active in erythroid cells. This would -include, but is not limited to, a globin gene promoter, such as the human alpha, beta, delta, 5 epsilon o zeta promoters, or a globin promoter from " anothe "species. It may, for example, be useful to utilize 'pig globin promoter sequences.- For example, as discussed in Section 10, infra, the use of the endogenous pig β globin gene control region, as ' 10 "contained Jn plasmid Pgem5/Pig pr(K) , deposited with the ATCC and assigned accession number 75371 and -having the sequence set forth in Figure 8, has been shown to operate particularly efficiently. The human alpha and beta globin genes may be placed under the 15 cdfttrol of different promoters, but, since it has 'been inferred that vastly different levels of glPbin chain 'production may result in lethality, it 'may be preferable to place the human alpha and beta globin gerϊes under the control of the same promoter sequence. ■ "o I 'order to avoid chain imbalance and/or titration of transcription factors due to constitutive 0-globin prdmoter activity in an inappropriate cell type, it is desirable to design a construct which leads to '" ' coordinate expression of human alpha and beta globiri' 25 genes at the same time in development and at quantitatively similar levels. -In one particular, non-limiting embodiment of the invention, a construct comprising the ααβ construct (also termed the "116" construct; Swansdn έt 30 al., 1992, Bio/Technol. 1^:557-559; see Figure 1A) may be utilized. Although this construct, when present as a transgene at high copy number, has resulted in deleterious effects in mice, it has been used to produce healthy transgenic pigs (see Example Section 3 6, infra) . In another particular, non-limiting embodiment of the invention, a construct comprising the αpβ sequence (also termed the "185" construct; see Figure IB) may be used. ' Such a construct has the advantage of placing both alpha and beta globin- encoding sequences under the control of the same 5 promoter (the alpha globin promoter) . In another particular, non-limiting embodiment of the invention, a construct coding for di-alpha globin like pόlypeptides may be introduced to form transgenic pigs that produce human hemoglobins 0 vith decreased dimerization and an increased half-life (WO Patent 9013645) ' . - In yet another particular, non-limiting embodiment of the invention, a construct comprising the human adult alpha globin and epsilon globin gene, 5 the pig beta globin gene control region and the human beta globin gene " (the "339' construct, see Figure 1R) may be used * <■ > ' ' -. ' Furthermore, the incorporation of a human or pig epsilon globin gene into " the construct may Ό facilitate the production of high hemoglobin levels. The pig epsilon globin ι gene may permit correct developmental regulation of the adult β globin gene. High levels of expression of introduced adult alpha globin gene(s) may 1 result in a chain imbalance problem 5 during intra-uterine development of a transgenic pig embryo (because an adult beta globin gene in the construct would not yet be expressed) thereby compromising the viability Of the embryo. By providing high levels of embryonic globins during 0 development, the viability of such embryos may be improved. The pig epsilon globin gene, as contained in plasmid pSaf/Pige, deposited with the ATCC and assigned accession number 75373, is shown in Figure 13. 5 ' The present invention, in further specific embodiments, provides for (i) the construct /Spa, in which the human alpha and beta globin genes are driven by separate copies of the human beta globin promoter (Figure 1C) ; ,(ii) the epfβpα construct, which comprises, human embryonic genes zeta and epsilon under the control of the epsilon promoter and both alpha and s beta genes ,under the control of the beta promoter (Figure ID) ; (iii) the fpeαp/3 construct, which comprises human embryonic genes zeta and epsilon under the control of the zeta promoter and both alpha and beta, genesiunder the control of the alpha promoter 1 0 Figure.rlE) ; (iv) the αp/3 construct carrying a mutation that results in an aspartic acid residue ^ at er, than an asparagine residue) at amino acid number 108 of β globin protein, to produce hemoglobin Yoshizuka (Figure IF, construct "294") ; (v) the apβ 5 construct carrying a mutation- that results in a lysine residue (rather than an asparagine residue) at amino .ac d number 108 of jβ-globin protein, to produce iiemoglobin. Presbyterian (Figure 1G, construct "293") ; > vil) the αp3(Δα) construct, coinjected with LCR a 0 which' comprises the human β-globin gene under the control of- the human α-globin promoter and a separate nucleic acid fragment comprising the human α-globin -, ^gene underι its own promoter (Figure 1H) ; (vii) the αpjβ Construct .carrying a mutation that results in a ,,25 cysteine residue (rather than a threonine residue) at amino acid number 134 of α-globin protein (Figure II) ; (viii) the αp/3 construct carrying a mutation that results in a serine residue (rather than a cysteine residue) at amino acid number 104 of the α-globin- 30 protein, .an alanine residue (rather than a cysteine residue) at amino acid number 93 of the jS-globin protein and a valine residue (rather than a cysteine residue) at amino acid number 112 of the 3-globin protein (Figure 1J) ; (ix) the αpδ construct, which -35 comprises the human adult α-globin promoter under its own promoter and the human 5-globin gene under the control of the human adult α-globin promoter (Fig. IK); (x) Construct αp£(Δα) coinjected with LCR α, which comprises the human <5-globm gene under the control of the human α-globin promoter and a separate nucleic acid fragment comprising the human α-globin 5 gene under its own promoter (Fig. IL) ; (xi) Construct LCR α coinjected with LCR eβ , which comprises the human α-globin gene under the control of its own ^ promoter and a separate nucleic acid fragment Comprising the human embryonic e-globin gehe and the ijj adult 0-globin gene under the control of their own promoters (Fig. 1M) ; (xii) the αpβ construct carrying a ftiutation that results in a methionine residue (rather than a lysine residue) at amino acid number 61 of the α-globin protein (Fig. IN); (xiii) the eaβ l£ Construct, which comprises the human embryonic epsilon gene, the human adult alpha globin gene and the human hdttlt beta globin gene linked in tandem from 5 , ~ to 3 1 (Fig. lO) ; (xiv) the aeβ construct, which -comprises the human adult alpha-globin gene, the human embryonic o epsilon globin gene and the human adult beta globin gene linked in tandem from 5 1 - to 3' (Fig. IP); (xv) the ύtaeβ construct, which comprises two Copies of the human adult alpha-globin gene, the human embryonic epώilon globin gene and the human adult beta globin 5 gene linked in tandem from 5'- to 3' (Fig. 1Q) ; (xvi) thfe tt€ ( p ' 8 j8p)3 construct, which comprises the human ' ^ adult alpha-globin gene, the human embryonic epsilon globin gene and the human adult beta globin gene under the control of the endogenous porcine adult beta - B globin promoter all linked in tandem from 5'- to 3* Fig. 1R) ; (xvii) the apβ construct carrying a-' mutation that results in a cysteine residue (rather than an aspartic acid residue) at amino acid number 75 of the α-globin protein (Fig. IS) ; (xviii) the apβ 5 construct carrying a mutation that results in an arginine residue (rather than a tyrosine residue) at amino acid number 42 at the α-globin protein (Fig. IT) ; (xvix) the LCR eβαα construct, which comprises the human embryonic epsilon -globin gene, the human adult beta globin gene and two copies of the human adult alpha-globin gene linked in tandem from 5 • - to 3' (Fig. 1U) ; (xx) the LCR eβa construct, which comprises the human embryonic epsilon globin gene, the human adult beta globin gene and the human adult alpha-globin gene linked in tandem from 5'- to 3 1 (Fig. IV) ; (xxi) the αp/3 construct carrying a mutation that results in a lysine residue (rather than a tyrosine residue) at amino acid number 42 of the a- globin protein (Fig. 1W) ; (xxii) the apβ construct carrying a mutation that results in an arginine residue (rather than a tyrosine residue) at amino acid number 42 at the α-globin protein and a glutamic acid residue (rather than an aspartic acid residue) at amino acid number 99 of the /3-globin prptein ' (Fig. lX); (xxiii) the αp/3 construct carrying a mutation that results in a lysine residue ' (rather than a tyrosine residue) at amino acid number 42 of the α- globin protein and a glutamic acid residue (rather than an aspartic acid residue) at amino acid number 99 of the 3-globin protein (Fig. 1Y) ; and (xxiv) the α p « e (P β βp)β construct comprising the pig epsilon globin gene and beta globin control region (constructs 426 and 427, Figure 14). In transgenic pigs expressing human hemoglobin three types of hemoglobin di ers are detectable: pig α/pig β , human α/human β , and hybrid human α/pig β . In certain embodiments of the invention, it may be desirable to decrease the amount of hybrid hemoglobin. Accordingly, the molecular basis for the formation of hybrid hemoglobin has been investigated using molecular modeling studies. Based on the information derived from these studies, the human alpha and beta globin structures can be modified to increase the level of human α/human β dimers (See Section 11.) , so that in further embodiments of the invention, constructs comprising the αp/3 sequence may be "modified to code for α or jS globin proteins carrying 'amino acid changes that will lead to increases in the level of human α/human β hemoglobin dimers irt transgenic pigs. The present invention, provides for constructs which encode human α globin and human β globin carrying one or more of the following mutations in the α globin molecule: (1) a Thr at position 30 instead of Glu; (ii) a Tyr at position 36 instead of Phe; (iii) a Phe instead of Leu at position- 106; (iv) a Ser or Cys instead of Val at position 107; and/or (v) a Cys instead of Ala at position 111. ■ In specific embodiments, the construct carrying such mutation(s) is the αpβ construct. The present invention, in further embodiments, provides for constructs which encode human α globin and human β globin carrying one or more of the following mutations in the β globin molecule: (1) a Leu instead of Val at > position' 33; (ii) a Val or lie instead of Cys at position 112; (iii) a Val or Leu instead of Ala at position*at position 115; (iv) a His instead of Gly at position 119; (v) a Met instead of Pro at position 125;r (vi)> an lie instead of Ala at position 128; - and/or (vii) a Glu instead of Gin at position 131; and/or (viii) a Glu instead of Gin at position 131. In specific embodiments, the construct carrying the mutation(s) is the αpβ construct. In further embodiments it may be desirable to include, in constructs, the untranslated 3* end of the pig beta globin gene as contained in plasmid pPig3'β (Figure 16) as deposited with the ATCC and assigned accession number 75372. (see, for example, construct 354 in Figure 12 and Figures 426 and 427 in Figure 14) . Such constructs may also be useful in the expression of non-globin protein in pig erythrocytes. In further embodiments, the pig beta globin control region depicted in Figures 8 and 9 may be used in constructs that encode non-globin proteins for the expression of said proteins in transgenic pig or other non-human erythrocytes. The recombinant nucleic acid constructs described above may be inserted into any suitable plasmid, bacteriophage, or viral vector for amplification, and may thereby be propagated using methods known in the art, such as those described in Maniatis et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y. In the working examples presented below, the pUC vector (Yanish- Perron et al., 1985, Gene 103-119) was utilized. The present invention further provides for isolated and purified nucleic acids comprising the pig adult beta globin promoter regulatory region, the_pig 3' beta globin region, and the pig epsilon globih? gene as comprised, respectively, in plasmids pgem5/PigSpr(K) (ATCC accession no. 75371), p igS 1 ^ (ATCC accession no. 75372), and pSaf/pige (k) (ATCC accession no. 75373) , respectively. Constructs may desirably be. linearized for preparation of transgenic pigs. Vector sequence may desirably be removed. 5.2. PREPARATION OF TRANSGENIC PIGS The recombinant constructs described above may be used to produce a transgenic pig by any method known in the art, including but not limited to, microinjection, embryonic stem (ES) cell manipulation, electroporation, cell gun, transfection, transduction, retroviral infection, etc. Species of constructs may be introduced individually or in groups of two or more types of construct. According to a preferred specific embodiment of the invention, a transgenic pig may be produced by the methods as set forth in Example Section 6, infra. Briefly, estrus may be synchronized in sexually mature gilts (>7 months of age,) by feeding a -orally active progestogen (allyl trenbolone, AT; 15 mg/gilt/day) for 12 to 14 days. On the last day of AT feeding all gilts may be given an intramuscular injection (IM) of prostaglandin F 2a (Lutalyse: 10 mg/injection.) at 0800 and 1600 hours. Twenty-four hours after the. last day of AT consumption all donor gilts may be .administered a single IM injection of pregnant mare-cserum gonadotropin (PMSG: 1500 IU) . Human chorionic gonadotropin (HCG: 750 IU) may be administered to all donors at 80 hours after PMSG. > , . Following AT withdrawal, donor and recipient gilts may be checked twice daily- for signs of estrus using a mature boar. Donors which' exhibited estrus within 36 hours following HCG administration may be bred at 12 and 24 hours after the, onset of. estrus using artificial and natural (respectively) , insemination. u->- < i Between 59 and 66 hours-after, the administration of HCG one- and two-cell ova may be surgically recovered from bred donors using the following procedure. General anesthesia may be induced by administering 0.5 mg of acepromazine/kg of bodyweight and 1.3 mg ketamine/kg of„ bodyweight via a peripheral ear vein. Following, anesthetization, the rep r oductive tract may be exteriorized following a miά-ventral laparotomy. A drawn glass cannula (O.D. 5 mm, length 8 cm) may be inserted into the ostium of the oviduct and anchored to ^ the infundibulum using a single silk (2-0) suture. Ovav may be flushed in retrograde fashion by inserting- a 20 g needle into the lumen of the oviduct 2 cm anterior to the uterotubal junction. Sterile Dulbecco's phosphate buffered saline (PBS) supplemented with 0.4% bovine serum albumin (BSA) may be infused into the oviduct and flushed 'toward the glass cannula. The medium may be collected into sterile 17 x 100 mm polystyrene tubes. Flushings may be transferred to 10 x 60 mm petri dishes and searched at lower power (50 x) using a Wild M3 stereomicroscope. All one- and two-cell ova may be washed twice in Brinster's Modified Ova Culture-3 medium (BMOC-3) supplemented, with 1.5% BSA and transferred to 50 μl drops pf BMOC-3 medium under oil. Ova may be stored at 38,°C under a 90% N 2 , 5% 0 2 , 5% C0 2 atmosphere until microinjection is performed. One- and two-rce,ll, ova may be placed in a Eppendorf tube (15 ova per >tube) containing 1 ml HEPES Medium supplemented with-1.5% BSA and centrifuged for 6 minutes at 14000 x g in order to visualize pronuclei in. one-cell and nuclei in two-cell ova. Ova may then be transferred to a 5 - 10 μl drop of HEPES medium under oil on a depression slide. Microinjection may be performed using <a Laborlux microscope with Nomarski optics and two Leitz micromanipulators. 10- 1700 copies of construct U (linearized at a concentration of about lng/μl of Tris-EDTA buffer) may be injected into one pronuclei in one-cell ova or both nuclei in two-cell ova. Microinjecfced, ova may be returned to microdrops of BMOC-3 medium under;oil and maintained at 38°C under a 90% N 2 , 5% C0 2 , 5% 0 2 atmosphere prior to their transfer to suitable recipients. Ova may preferably be transferred within 10 hours of recovery. Only recipients which exhibit estrus on the same day or 24 hours later than the donors may preferably be utilized for embryo transfer. Recipients may be anesthetized as described earlier. Following exteriorization of one oviduct, at least 30 injected one-and/or two-cell ova and 4-6 control ova may be transferred in the following manner. The tubing from a 21 g x 3/4 butterfly infusion set may be connected to a 1 cc syringe. The ova and one to two mis of BMOC-3 medium may be aspirated into the tubing. The tubing may then be fed through the ostiu of the oviduct until the tip reaches the lower third, or isthmus of the oviduct. The ova may be subsequently - expelled as the tubing is slowly withdrawn. - • • ■ ■ The exposed portion of the reproductive tract may be bathed in a sterile 10% glycerol-0.9% saline solution and returned to the body cavity * - The connective tissue encompassing the linea alba,, the fat and the skin may be sutured as three separate layers. An uninterrupted Halstead stitch may be used to,.close the lina alba. The fat and skin may be closed using a simple continuous and mattress stitch, respectively. A topical antibacterial agent (e.g. Furazolidon.e) -may then be administered to the -incision area. - _ Recipients may be penned in groups -of. about four and fed 1.8 kg of a standard 16% crude prpteiη corn-soybean pelleted ration- Beginning on day 18 (day 0 = onset of estrus) , all recipients may be , checked daily for signs of estrus using a mature boar. 0n ctay 35, pregnancy detection may be performed using ultrasound. On day 107 of gestation recipients may be transferred to the farrowing suite. In order to ensure attendance at farrowing time, farrowing may be induced by the administration of prostaglandin F 2a (10 mg/injection) at 0800 and 1400 hours on day 112 of gestation. In all cases, recipients•<may be expected to farrow within 34 hours following,PGF2a administration. Twenty-four hours after birth, all, iglets may be processed, i.e. ears notched, needle teeth clipped, 1 cc of iron dextran administered, etc. A tail biopsy and blood may also be obtained from each pig. Pigs produced according to this method are described in Example Section 6, infra, and are depicted in Figure 2. Such pigs are healthy, do not appear to be anemic, and appear to grow at a rate comparable to that of their non-transgenic littermates. Such pigs may transmit the , ransgene to their offspring. Pigs having certain characteristics may be especially useful for the production of human hemoglobin; such pigs, examples of which follow, represent preferred, non-limiting, specific embodiments of the , invention. According to one ,preferred specific embodiment of the invention, a transgenic pig contains at least twenty copies of a globin transgene. According to a second preferred specific embodiment, the P 50 of whole blood of a transgenic pig according to the invention is increased by at least ten percent over the P 50 ,of the whole blood -of a comparable non-transgenic pig, aking into consideration factors such as altitude, oxygen concentrations, pregnancy,, the presence of mutant hemoglobin, etc. Thus, the present invention provides for a non-pregnant transgenic pig that carries and expresses a human globin transgene in which the P 5() of whole blood of the transgenic pig is at least ten percent greater than the P s0 of .whole blood of a comparable non-pregnant non—transgenic pig at the same altitude. , , • In other preferred specific embodiments, the present invention provides for a transgenic pig in which the amount of human globin produced relative to total hemoglobin is at least two. percent, more preferably at least five percent, and most preferably at least ten percent. Section 6, infra, describes transgenic pigs which serve as working examples of preferred, non- limiting, specific examples of the invention. 5.3. PREPARATION OF HUMAN HEMOGLOBIN AND ITS SEPARATION FROM PIG HEMOGLOBIN The j presen inyention provides for a method for producing humarf hemoglobin comprising introducing a transgene or transgeheέ encoding huihan hemoglobin, such as a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the gertetic material of a pig so as to create a transgenic pig that expresses human hemoglobin in at least 'Sθτtι6 of its blood cells. The present invention also provides for a method of producing htimart hemoglobin comprising (i) introducing a human alpha globin and a human beta globin gene, under the control of a suitable promoter or promoters, into the genetic material of a pig so as to create a transgenic pig that expresses human hemoglobin in" " at least sdme f ' its red blood cells; (ii) collecting red blood cells from the transgenic pig; (iii) releasing the 1 contents of the collected red blood cells to form a lys'ate; (iv) Subjecting the lysate of the red blood cells to a purification procedure that substantially separates human hemoglobin Jrrom pig hemoglobin; and (v) collecting the fractions that contain purified human hemoglobin. Such fractions may be identified by isoelectric focusing in parallel with appropriate standards. In a preferred embodiment of the invention, human hemoglobin may be separated from pig hemoglobin by DEAE anion exchange column chromatography. In order to prepare human hemoglobin from the transgenic pigs described above, red blood cells are obtained from the pig using any method known in the art. The red blood cells are then lysed using any method, including hemolysis in a hypotonic solution such as distilled water, or using techniques as described in 1981, Methods in Enzymology Vol. 76, and/or tangential flow filtration. For purposes of ascertaining whether human hemoglobin is being produced by a particular transgenic pig, it may be useful to perform a small- scale electrophoretic analysis of the hemolysate, such as, for example, isoelectric focusing using standard techniques.c Alternatively, or for larger scale purification, human hemoglobin may be separated from pig hemoglobin using ion exchange chromatography. Surprisingly; -as discussed in Section 7, supra, human hemoglobin was observed to readily separate from pig hemoglobin using ion exchange chromatography whereas mouse he pglobin and human hemoglobin were not separable by such methods. Any ion exchange resin known in the art or to be developed may be utilized, including, but not limited to, resins comprising diethylaminoethyl, Q-Sepharose, QCPI (I.B.F.) Zephyr, Spherodex, eptiola, carboxymethylcellulose, etc. provided that the resin results in a separation of human and pig hemoglobin comparable to that achieved using DEAE resin. According to a specific, nonlimiting embodiment of the invention, in order to separate human from pig hemoglobin (including human/pig hemoglobin hybrids) to produce substantially pure human hemoglobin, a hemolysate of transgenic pig red blood cells, prepared as above may be applied to a DEAE anion exchange column equilibrated with 0.2 M glycine buffer at Ph 7.8 and washed with 0.2 M glycine Ph 7.8/5 Mm NaCl, and may then be eluted with a 5-30 Mm NaCl gradient, or its equivalent (see, for example, Section 9 infra) . Surprisingly, despite about 85 percent homology between human and pig globin chains, human and pig hemoglobin separates readily upon such treatment, with human hemoglobin eluting earlier than p g hemoglobin. Elution may be monitored by optical density at 405 nm and/or electrophoresis of aliquots taken from serial fractions. Pig hemoglobin, as well as tetrameric hemoglobin composed of heterodimers formed between pig and human globin chains, may be separated from human hemoglobin by this method. Human hemoglobin produced in a< transgenic pig and separated from pig hemoglobin by this method has an oxygen binding capability similar to that of native human hemoglobin. • > According to another specific, non-limiting embodiment of the invention, human hemoglobin may be separated from pig hemoglobin (including human/pig hemoglobin hybrids) using QCPI ion exchange resin as follows: About 10 mg of hemoglobin prepared from transgenic pig erythrocytes may be diluted in 20ml of Buffer A (Buffer A, F lOmM Tris,^ 20mM Glycine Ph 7.5). This 20ml sample may then be loaded at a flow rate of about 5ml/min onto a QCPI' Column (10 ml) which has been equilibrated with Buffer A. The column may then be washed with 2 volumes of Buffer A,„and then with 20 column volumes of a 0-50mM NaCl gradient (10 column volumes of Buffer A + -1-0 column volumes of lOmM Tris, 20mM Glycine, 50mM NaCl Ph 7.5) or, alteratively, 6 column volumes of lOmM Tris > 20mM Glycine, 15mM NaCl, pH 7.5, and the O.D. 28u absorbing material may be collected in fractions to yield the separated hemoglobin, human hemoglobin being identified, for example, by isoelectric focusing using appropriate standards. The QCPI column may be cleaned by elution with 2 column volumes of lO M-Tris, 20mM Glycine, IM NaCl, pH 7.5. For certain mutant hemoglobins, it may be desirable to utilize a modified purification procedure. Accordingly, for the separation of Hb Presbyterian from pig Hb, a procedure as described in Example Section 12.1, infra, may be used, and for separation of Hb Yoshizuka, a procedure as described in Example Section 12.2, infra, may be used. ■ 5.4-. PREPARATION OF HUMAN/PIG HYBRID HEMOGLOBIN The present invention also provides for essentially purified and isolated human/pig hybrid hemoglobin, in particular human α/pig β hybrid hemoglobin. Pig α/human has not been observed to form either in vitro in reassociation experiments or In vivo in transgenic pigs. ->The present invention provides for hybrid hemoglobin and its -use as a blood substitute, and for a pharmaceutical composition comprising the essentially purified and isolated human/pig hemoglobin hybrid in a suitable pharmacological carrier. Hybrid hemoglobin may be prepared from transgenic pigs,, -as described herein, and then purified.by chromatography, i munoprecipitation, or .any other method known to the skilled artisan. The use of isoelectric focusing to separate out hemoglobin hybrid is shown (in Figures 3 and 5. Alternatively, hybrid hemoglobin may be "prepared using nucleic acid constructs that comprise both human and pig globin sequences which may then be expressed in arty suitable microorganism, cell, or transgenic animal. For example, a nucleic acid construct that comprises the human α and pig β globin genes under the control of .a suitable promoter may be expressed to result in hybrid hemoglobin. As a specific example, human ,ς_ globin and pig β globin genes, under the control of cytomegalovirus promoter, may be transfected into a mammalian cell such as a COS cell, and hybrid hemoglobin may be harvested from such cells. Alternatively, such constructs may be expressed in yeast or bacteria. It may be desirable to modify the hemoglobin hybrid so as to render it non-im unogenic, for example, by linkage with polyethylene glycol or by encapsulating the hemoglobin in a membrane, e.g. in a liposome. 6. EXAMPLE: GENERATION OF TRANSGENIC PIGS THAT PRODUCE HUMAN HEMOGLOBIN 6.1. MATERIALS AND METHODS .(3.1.1. NUCLEIC ACID CONSTRUCTS Constructs 116 (the αα/3 construct) , 185 (the αpβ construct), 263 (the αp<5 construct) 339, 293 and 294 were microinjected into pig ova as set forth below in order to produce transgenic pigs. 0 6. ' l.y. PRODUCTION OF TRANSGENIC PIGS Estrus was synchronized in sexually mature gilts ( 7 months of age) by feeding an orally active progestoge'n (allyl trenbolone, AT: 15 mg/gilt/day) for 12 to 14 days. On the last day of AT feeding all 5 gilts received an' intramuscular injection (IM) of prostagl-andin F 2a (Lutalyse: 10 mg/injection) at 0800 and ,J600. , T/enty-four hours after the last day of AT consumption all donor gilts received a single IM injection of pregnant mare serum gonadotropin (PMSG: 0 1500 IU) 'Human choriortic gonadotropin (HCG: 750 IU) was administered to all donors at 80 hours after PMSG. Following AT withdrawal, donor and recipient gilts were checked twice daily for signs of estrus using a mature boar. Donors which exhibited estrus 5 within 36 hours following HCG administration were bred at 12 and 24 hours after the onset of estrus using artificial and natural (respectively) insemination. Between 59 and 66 hours after the administration of HCG, one- and two-cell ova were Q surgically recovered from bred donors using the following procedure. General anesthesia was induced by administering 0.5 mg of acepromazine/kg of bodyweight and 1.3 mg ketamine/kg of bodyweight via a peripheral ear vein. Following anesthetization, the 5 reproductive tract was exteriorized following a mid- ventral laparotomy. A drawn glass cannula (O.D. 5 mm, length 8 cm) was inserted into the ostium of the oviduct and anchored to the infundibulum using a single silk (2-0) suture. Ova were flushed in retrograde fashion by inserting a 20 g needle into the 5 lumen of the oviduct 2 cm anterior to the uterotubal junction. Sterile Dulbecco's phosphate buffered ι saline (PBS) supplemented with 0.4% bovine serum J, albumin (BSA) was infused into the oviduct and flushed I toward the glass cannula. The medium was collected IJ J O .into sterile 17 x 100 mm polystyrene tubes. Flushings, were transferred to 10 x 60 mm petri dishes and J < searched at lower power (50 xj using a Wild M3 < stereomicroscope. All one- and two-cell ova were washed twice in Brinster's Modified Ova Culture-3 - , medium (BMOC-3) supplemented with 1.5% BSA and transferred to 50 μl drops of BMOC-3 medium und^r oil.' Ova / were stored at 38°C under a 90% N 2 , 5% 0 2 , 5%w 'C0 2 I atmosphere until microinjection was performed. . One- and two-cell ova were placed in an " i r 20 Eppendorf tube (15 ova per tube) containing 1 ml 'HEPES Medium supplemented with 1.5% BSA and centrifuged for 6 minutes at 14000 x g in order to visualize pronuclei 1 in one-cell and nuclei in two-cell ova. Ova were then transferred to a 5 -10 μl drop of HEPES medium under 25 °il on a depression slide. Microinjection was performed using a Laborlux microscope with Nomarski optics and two Leitz micromanipulators. 10-1700 copies of construct DNA (lng/μl of Tris-EDTA buffer) were injected into one pronuclei in one-cell ova or 30 both nuclei in two-cell ova. Microinjected ova were returned to microdrops of BMOC-3 medium under oil and maintained at 38°C under a 90% N 3 , 5% C0 2 , 5% 0 2 atmosphere prior to their transfer to suitable recipients. Ova were 35 transferred within 10 hours of recovery. Only recipients which exhibited estrus on the same day or 24 hours later than the donors were utilized for embryo transfer. Recipients were anesthetized as described earlier. Following exteriorization of one oviduct, at least 30 injected one- and/or two-cell ova and 4-6 control ova were transferred in. the following manner. The tubing from a 21 g x 3/4 butterfly ( infusion set was connected to a 1 cc syringe. ' , The ova and one to two mis of BMOC-3 medium were aspirated into the tubing. The tubing was then fed through the ostium of the oviduct until the tip reached the lower third or isthmus of the oviduct. The ova were subsequently expelled as the tubing was slowly withdrawn. ' The exposed portion of the reproductive tract was bathed.in a sterile 10% glycerol-0.9% saline solution and returned to the body cavity. The connective tissue encompassing the linea alba, the fat and the skin,were sutured as three separate layers. An uninterrupted Halstead stitch was used to close the Una alba: ϊhe fat and skin were closed using a simple continuous and mattress stitch, respectively. A topical antibacterial agent (Furazolidone) was then administered to the incision area. Recipients were penned in groups of four and fed 1.8 kg of a standard 16% crude protein corn- soybean pelleted ration. Beginning on day 18 (day 0 = onset of estrus) , all recipients were checked daily for signs of estrus using a mature boar. On day 35, pregnancy detection was performed using ultrasound. On day 107 of gestation recipients were transferred to the farrowing suite. In order to ensure attendance at farrowing time, farrowing was induced by the administration of prostaglandin F 2j (10 mg/injection) at 0800 and 1400 hours on day 112 of gestation. In all cases, recipients farrowed within 34 hours following PGF2a administration. Twenty-four hours after birth, all piglets were processed, i.e. ears were notched, needle teeth clipped, 1 cc of iron dextran was administered, etc. A tail biopsy and blood were also obtained from each pig. ' 6.2. RESULTS AND DISCUSSION Of 3566 injected ova, thirteen transgenic 'pigs that expressed human hemoglobin were born, two of which died shortly after birth due to normal breeding- related ' incidents completely unrelated to the fact that they were transgenic pigs (Table I) . The remaining "ϊi appeared to be healthy. A photograph of one transgenic pig is presented in Figure 2. Profiles of the' pigs ' and of the percent "authentic" and "hybrid" human hemoglobin ("HB") produced are set fdrth in Table II, infra. Total hemoglobin Was calculated as the sum of human aβ plus one-half of 'the human " a pig β hybrid. Figure 3 presents the results Of isoelectric focussing and triton acid urea gels of hembg ' lobin produced by three of these pigs (numbers 12-1, 9-3, and 6-3) which demonstrate the expression of human alpha and beta globin in these animals. TABLE I Efficiency of Transgenic Pig Production Human Hemoglobin Gene Construct(s) Total After Parameter 22 Trials Total Ova Collected 8276 Total # Fertilized 7156 Total # Injected 3566 # Injected Ova Transferred 3566 # Control Ova Transferred 279 # Recipients Used 104 # Pigs Born (Male, Female) 208,332 # Transgenic (Male, Female) 8,5 (0.36) 3 ' > # Expressing 13 -, - > I a Proportion of injected ova which developed into transgenic pigs (13 transgenics/3566 injected ova) . TABLE II FOUNDERS Table III presents the profiles of offspring of pig number 9-3, which shows that the Fl generation of transgenic pigs are capable of expressing hemoglobin. Of note, none of the offspring of pig nttmbet 6-3 were found to be transgenic, possibly due to the absence of transgene in the animal's reproductive tissue. , - Table IV presents hemoglobin expression data of, offspring of pig 38-4 carrying the "185" construct (the "αpjS" construct; see Figure IB) . Table V pifesents a summary of the profiles of offspring of pig number 38-4 in which a large percentage (37.1%) of Offspring were positive for expression of human hemoglobin indicating germ line transmission of tile transgene. Figure 19 presents the results of isoelectric focussing which demonstrates the levels of hemoglobin expression in representative transgene positive 38-4 offspring. TABLE III Fl (OFFSPRING) OF PIG 9-3 *9-3-2 died the,day after birth- '> , - -, I Λ -. \- i TABLE IV 10- 15 20 25 TABLE IV (CONT'D) 10 15 20 25 TABLE V 38-4 BREEDING SUMMARY 10 FOUNDER LITTERS PIGLETS PIGS/LITTER TRANSGENIC FREQUENCY AVG. AUTHENTIC HbA 38-4(M) 34 318 9.4 1 18 37.1 % 6.2% AUTHENTIC HUMAN HB AUTHENTIC HUMAN HB is MALES EXPRESSION LEVEL FEMALES EXPRESSION LEVEL 59 5.7% 59 6.8% 20 25 The birth weights .of the transgenic pigs have been approximately equivalent ϊto the birth weights of their non-transgenic littermates. As the transgenic pigs matured, their weights remained comparable to the weights of control animals. 7. EXAMPLE: SEPARATION OF HUMAN HEMOGLOBIN FROM PIG HEMOGLOBIN BY DEAE CHROMATOGRAPHY 7.1. MATERIALS A'ND METHODS 7.1.1. PURIFICATION BY DEAE CHROMATOGRAPHY For purification, ' red blood cells were collected by centrifugation of 5000 rpm for 3 minutes in an eppendorf microcentri ug'e and washed three times with an equal volume (original blood) of 0.'9% NaCl. Red cells were lysed with ' J .' S Volumes deionized H 2 0, centrifuged at 15,000 rpm, and the supernatant was fractionated by anion exchange chromatography. DEAE cellulose chromatography (DE-SE " manufactured by Whatman, Ltd.) was performed according to W. A. Schroeder and T. H. J. Huisman "The Chromatography of Hemoglobin", Dekker, New York, pp. 74-77. The 0.25 ml red cell hemolysate described, above was applied to 1 cm x 7 cm DE-52 column Cpre-.eguilibrated in 0.2 M glycine Ph 7.8 and "Was washed with 5, column volumes of 0.2 M glycine Ph 7.8/5..Mm NaCl. Hemoglobins were eluted with a 200 ml 5 30 mM NaCl/0.2 M glycine pH 7.8 gradient. To complete elution of pig hemoglobin, an additional 50 to 100 ml of 30 mM CaCl/glycine pH 7.8 was added to the column. Elution of hemoglobin was monitored by absorbance of 415 >mM and by IEF analysis of column fractions. 7.1.2. REASSOCIATION OF GLOBIN CHAINS Reassociation of globin chains was performed essentially as described in Methods in Enzymol. 2^:126-133. 25 lambda of pig blood, 25 lambda of human bloodyor a 25 lambda mixture of 12.5 lambda human blood and , 12.5 lambda pig blood were treated as follows. The blood was pelleted at a setting of 5 on microfuge for 2 minutes, then washed three times with 100 lambda 0.9 percen > NaCl. The cells were lysed with 50 lambda H 2 Q, then spun -at high speed to confirm lysis. 50 lambda of the lysed cells was then combined with 50 lambda c 0,2 M ^Ja Acetate pH 4.5, put on ice and then incubated in a cold room overnight. After adding 1.9 ml 0.1 M NaH 2 P0 4 4, . pH 7.4 each sample was spun in centricon tubes at 4°C and 5K until about 0.5 ml remained. Then.l ml of 0.1 M NaH 2 P0 4 pH 7.4 was added and spun through at_about 5K until about 0.2 ml volume yras left. -The hemoglobin was then washed from the walls of the pentricon tube, an eppendorf adaptor was attached, and -a table- top microfuge was used to remove each sample from its centricon tube. The samples were then analyzed by isoelectric focusing. „ 7.2. RESULTS AND DISCUSSION 7.2.1< HUMAN AND PIG HEMOGLOBIN WERE SEPARATED FROM A HEMOLYZED MIXTURE OF HUMAN AND PIG BLOOD Egual proportipns of human and of pig blood were mixed and lysed, and the resulting hemolysate was subjeoted to DEAE chromatography as described supra. As shown in Figure 4A, pig hemoglobin separated virtually completely from human hemoglobin. This complete separation is surprising in light of the structural similarity between human and pig hemoglobin; pig and human alpha globin chains are 84.4 percent homologous and pig and human beta globin chains are 84.9 percent homologous. It is further surprising because, as shown in Figure 4C, when human and mouse blood was mixed, hemolyzed, applied to and eluted from a DEAE column according to methods set forth in Section 7.1.1., supra, human and mouse hemoglobin '-were not observed to separate despite the fact that mouse and human alpha globin chains are about 85.8 percent homologous and mouse and human beta globin chains are 80.1 percent homologous. The ease of 5 separation of human and pig hemoglobin on DEAE resin appears to be both efficient and economical. Interestingly, the order of elution of the proteins .from the anion exchange column was not as .expected. s Based on the relative pi's of the proteins 10 as deduced from the IEF gels, the predicted order of elution would be first the hybrid (human α/pig β) , followed by the authentic human α/human β . The last prptein tp elute from the anion exchange column then τ would be the endogenous pig α/pig β protein. However, • 15 under, all the conditions currently attempted the order of elution was altered such that the human hemoglobin was the first to elute. The second peak was an enriched fraction of the hybrid followed very closely by the pig hemoglobin. 20 7.2.2. HUMAN AND PIG HEMOGLOBIN AND HUMAN/PIG HETEROLOGOUS HEMOGLOBIN WERE SEPARATED FROM HEMOLYSATE PREPARED FROM A TRANSGENIC PIG Blood from transgenic pig 6-3 (as described 25 in Section 6, supra) was lysed by hypotonic swelling and the resulting hemolysate was subjected to DEAE chromatography as described supra. As shown in Figure 4B, human hemoglobin was separated from pig hemoglobin and from human α globin/pig beta globin heterologous 30 hemoglobin. As shown in Figure 4D, human hemoglobin was substantially purified by this method. 7.2.3. PIG ALPHA GLOBIN/HUMAN BETA GLOBIN 35 HETEROLOGOUS HEMOGLOBIN DOES NOT APPEAR TO FORM BASED ON REASSOCIATION DATA Heterologoμs association between pig alpha globin and human beta globin chains has not been c detected in hemolysates obtained from human hemoglobin-expressing transgenic pigs. It was possible, however, that this observation could be explained by relatively low levels of human beta globin expression. Alternatively, association between pig alpha globin and human beta globin may be chemically unfavorable. In order to explore this possibility, reassociation experiments were performed in which pig and human hemoglobin were mixed, dissociated, and then the globin chains were allcwed tp reasspciate. As shown in the isoelectric focusing gels depicted in Figure 5, although pig α/pig β , human α/human β , and human α/pig β association was observed, no association between pig α globin and human β globin appeared to have occurred. Therefore the pig α/human β heterologous hemoglobin should not be expected to complicate the purification of human hemoglobin from transgenic pigs. EXAMPLE: SEPARATION OF HUMAN HEMOGLOBIN FROM PIG HEMOGLOBIN BY QCPI CHROMATOGRAPHY 8.1. MATERIALS AND METHODS Clarified hemolysate from transgenic pig 6-3 13mg/ml; Buffer A: lOmM Tris, 20mM Glycine pH 7.5; Buffer B: lOmM Tris, 20mM Glycine, 15 mM NaCl pH 7.5; Buffer C: lOmM Tris, 20mM Glycine, IM NaCl pH 7.5; Buffer D: lOmM Tris, 20mM Glycine, 50 mM NaCl pH 7.5; QCPI column 10ml Equilibrated in Buffer A; Trio purification system. lQmg of hemoglobin prepared from transgenic pig 6-3 was diluted in 20ml Buffer A. 20ml of sample was loaded at a flow rate of 5ml/min onto the QCPI column, and washed with 2 column volumes of Buffer A. The column was then washed with 20 column volumes of a 0-50mM NaCl gradient. (10 column volumes Buffer A'+ 10 Colum ' volumes of Buffer D) and the O.D. 2g0 absorbing material was collected. The column was then cleaned -with 2 column volumes of Buffer C, - and then fe-equilibrated with 2 column volumes of Buffer A.'■•' ■ ' > ■ • ' 8.2.' RESULTS '' '•• ' " Analysis Of the UV trace (peak vs. volume of gradient)" ' (Fig.' -6) 'revealed that the human hemoglobin was eluted at 15 M-NaCI-. Subsequent purifications have been -performed'utilizing " the same protocol as above, only using'6 column volumes of Buffer B (15mM NaCl) to elute the human hemoglobin rather than the gradient. In addition, non-transgenic pig chromatographed by this method does not elute from the QCPI with Buffer B, while native human hemoglobin does. The protein that eluted at 15mM NaCl was analyzed on the Resolve isoelectric focussing system and found to be essentially pure of contaminating pig hemoglobin or hybrid hemoglobin. 9. EXAMPLE: HUMAN ALPHA/PIG BETA GLOBIN HYBRID HEMOGLOBIN EXHIBIT INCREASED P<„ As shown in Tables II and III, supra. transgenic pigs of the invention were all found to produce significant amounts of human α/pig β globin hybrid hemoglobin (the pig α/human β hybrid was not observed) . Significantly, pigs that expressed higher percentages of hybrid also appeared to exhibit elevated P 5ϋ values for their whole blood (Figure 7) . 10. EXAMPLE: ENHANCED EXPRESSION USING PIG BETA GLOBIN REGULATORY SEQUENCES The 339 construct (Figures 1R and 12) containing the pig adult beta globin gene promoter region (Figure 8) , was used to prepare transgenic pigs according to the method set forth in Section 6.1.2. supra. Figure 15 depicts an isoelectric focusing gel analysis of hemoglobin produced by pig 70-3; equal amounts of hemoglPbin from transgenic pig 6-3, carrying the 116 construct (Figure 1A) and human hemoglobin are run in adjacent lanes for comparison. As indicated by the brighter bands observed in the lane containing pig 70-3 hemoglobin at positions correspbryjling to human and hybrid hemoglobins (relative^to the lane containing pig 6-3 hemoglobin) , the amount of human hemoglobin produced by pig 70-3 is greater?than the amount produced by pig 6-3. It has been calculated that 38 percent of the total hemoglobin produced by pig 70-3 is human hemoglobin, whereas^ ϊ) percent of total hemoglobin produced by pig r C 6-3^is efauStan hemoglobin (see Table II and Section 6.2. supra, -Tor data and calculations) . This suggests that .> F> - • . - _ the ∞ pig, beta globin promoter region is more efficient thajv the human beta globin promoter in transgenic pigs. 7 -^ 7 •' In a separate series of experiments, two more transgenic pigs, expressing human hemoglobin, •.: 'x, were obtained using construct "339" (pigs 80-4 and 81- 3) (FIG.17). Human hemoglobin levels in these transgenic pigs was determined by running isoelectric focussing gels and densito etric scanning of the individual bands (FIG. 18) . As indicated in Figure 17, both pig 70-3 and pig 80-4 expressed high levels of authentic human hemoglobin. To obtain the copy number of transgenes, genomic DNA (isolated from the tail) was digested with EcoR I and a Southern Blot was performed. The probe used was a 427 bp Ncol/Bam HI fragment of human beta globin gene containing the first exon, first intron and part of the second exon. 11. EXAMPLE: MOLECULAR MODELING OF PIG HEMOGLOBIN AND THE α, /3, INTERFACE OF A HYBRID BETWEEN PIG β AND HUMAN a GLOBIN It has been found that the amount of hybrid human α/pig β hemoglobin often exceeds the amount of human hemoglobin. The molecular basis of this observation has been investigated using molecular , modeling and molecular biology. The model structure of the hybrid molecule is based on the known ^ t structures of human hemoglobins and the strucjtural homology between the human and pig structures (A.M. Lesk, 1991, Protein Architecture: A Practical Approach, Oxford University Press, N.Y.). The pig and hybrid hemoglobin structures were modeled using the following four steps: (1) hydrogen atoms were added to the X-ray model and their positions modified using energy minimization; (2) amino acid residue replacements were introduced to model the ^ r 9 e pig and hybrid structures (no chain alignment was necessary) ; (3) the side chain positions of these modified residues were energy minimized; and (4) 'the result was visually examined and found to be sound. The modeled structures are shown in Figure.20. Detailed examination of all the relevant contacts indicated striking differences at several residues. For example, at position (3112 the human hemoglobin has a cysteine residue but the hybrid has a valine residue. The valine is in apparent closer contact (arrow in FIG. 20) with the opposing subunit, and thus may be more effective in stabilizing the α, /3, interface (FIG. 21) . The effect of amino acid substitutions at the α r jS, interface on -"the hydrophobic and polar interactions as predicted by HINT are shown in TABLE VI. HINT is software from Virginia Commonwealth University Licensed from Medical College of Virginia, Richmond, Virginia that can analyze the positive and negative scores as determined by attractive and repulsive interactions known from experimental physical chemistry measurements. TABLE VI represents the differences between the unmodified dimer and the one with the specified replacement. TABLE VII has the same format as TABLE VI with the following two exceptions: (l) as each replacement is added, the previous one(s) are kept, and (2) the reported difference is a comparison between the current dimer and the one reflected in the preceding row. As the subsequenlJal changes are " made, the predicted attractive forces at the interface increase. If each column is summed up the total difference between the unmodified dimer and the one with seven changes is obtained. The sums are +1340 for hydrophobic and +660 for' polar. TABLE VI Effect of amino acid replacements at the αl/3l interface 25 12. EXAMPLE: EXPRESSION OF GENETICALLY MODIFIED HEMOGLOBINS IN TRANSGENIC ANIMALS Of the known human hemoglobin variants, _ about two dozen exhibit a lower oxygen affinity, which could -be advantageous in clinical applications. While many of these mutants result in unstable hemoglobin molecules, several variants have desirable biochemical properties and can be used for the generation of blood 0 substitutes using recombinant•• DNA technology. Transgenic pigs expressing two of these variants, Hb Presbyterian (108 Asn→Lys, Fig. 1G) and Hb Yoshizuka (108 Asn→Asp, Fig. IF) have been produced and purification and characterization of the expressed 5 human globins is described below. 12.1. PURIFICATION AND CHARACTERIZATION OF Hb PRESBYTERIAN The amino acid substitution generated in Hb Presbyterian ( l08 Asn→Lys) results in the comigration of Hb Presbyterian with the hybrid (hαp/3) hemoglobin on isoelectric focussing gels. Based on previous results with the purification of human hemoglobin from hybrid and porcine hemoglobins and the more positive nature of the Hb Presbyterian it should be easier to purify this variant hemoglobin on an anion exchange resin. Approximately 500 ml of blood was obtained from the transgenic pig 57-10. The blood was washed several times with isotonic saline and then lysed by hypotonic swelling in water. The cell membranes were removed by centrifugation at 10000 xg to yield a final hemoglobin concentration of about 100 mg/ml. Hb Presbyterian was purified from the hybrid and porcine hemoglobins as follows: 1-2.5 g of hemolysate was loaded onto an XK 50/30 column packed with 450 ml of Biorad Macroprep High Q resin equilibrated with 10 mM Tris-Cl and 20 mM Glycine at pH 8.1 (Buffer A) . The proteins were eluted at a flow rate of 10 ml/min with a linear salt gradient of 9-16% Buffer B (Buffer A containing 250 mM NaCl) over 3000 ml. ,. , The initial peak was thought to be Hb Presbyterian followed by the co-elution of the hybrid aήd'pόrcine hemoglobins (FIG. 20) . To confirm the identity of the first peak as Hb Presbyterian and not the hybrid hemoglobin, a sample of the protein was run on Reversed Phase HPLC (FIG. 21) . The initial peak from the anion exchange column was Hb Presbyterian with the α-chains eluting at the same time as normal human α-chains and the 0-chains eluting slightly faster than normal human -chains. This was also found to be an excellent way of determining if porcine hemoglobin was contaminating the column fractions. Using this purification procedure and the analysis on HPLC the recombinant Hb Presbyterian derived from the transgenic pig 58-10 was judged to be greater than 95% pure. Purified Hb Presbyterian was dialyzed against 50 mM HEPES and 100 mM NaCl at pH 7.4 and oxygen equilibrium curves determined using a Hemox Analyzer (TCS Products, Southampton, PA). The Hemox Analyzer was modified to allow analog to digital data conversion for ease of oxygen binding calculations. Under these conditions the Hb Presbyterian had a P 50 of 25.8 mmHg (Hill Coefficient n=2.3) versus 13.3 mm Hg (n=2.9) for Hb A indicating that the Hb Presbyterian bound oxygen with lower affinity than native Hb. Preliminary results to determine the Bohr Effect (Influence of pH on the oxygen affinity) indicated a normal Bohr effect for Hb Presbyterian (FIG. 22) . 12.2. PURIFICATION AND CHARACTERIZATION OF Hb YOSHIZUKA Blood samples taken from the transgenic pigs expressing Hb Yoshizuka (68-3 and 68-2) were treated 5 essentially the same as described above.'^ The final concentration of the hemolysate was ^approximately 100 mg/ml. The purification of the protein required a slightly different strategy, however^ A sample of hemolysate from 68-3 (about 10 mg) was loaded onto an 10 HR 10/30 Biorad Macroprep High Q resin column equilibrated with 10 mM Tris-Cl and 20 mM Glycine at pH 8.7 (Buffer A). The hemoglobins were eluted at 2.5 mls/min with a 5-30% linear gradient -of Buffer B (Buffer A plus 250 mM NaCl) over 500.ml (FIG. 23). 15 Fractions were collected and analyzed by IEF to assess purity which was determined to -be about 75%ιor better. 13. DEPOSIT OF MICROORGANISMS The following plasmids were deposited .with 20 the American Type culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, Maryland 20852 on December 2, 1992. ' * < >-r, plasmid containing , accession , no. psaf/pige(k) pige 1 globin gene > 75371 '25 pGem5/Pig/3pr(K) pig adult β globin 75372 gene regulatory region pPig3'j8 3' end of pig 75373 β globin gene ; 30 Various publications are cited herein which are hereby incorporated by reference -in their entirety. 35 International Application No: PCT/ International Application No: PCT/ • j , , Form PCT RO/134 (cont.) American Type Culture Collection 12301 Parklawn Drive Rockville, MD 10582 US Accession No. Date of Deposit 75372 December 2. 1992 75373 December 2. 992

Claims: WHAT IS CLAIMED IS: - . . 1. A transgenic pig comprised of the DNA seguences encoding human alpha globin and human beta globin operably linked to promoter elements where human hemoglobin is produced in at least some of the red cells of said pig and in which the nucleic acid construct is the 426 construct - s depicted in Figure 14. - ,. , 2. A transgenic pig comprised ,of the DNA sequences encoding human alpha globin and h man beta globin operably linked to promoter elements where human hemoglobin is produced in at least some of the red cells of said pig and In which the nucleic acid construct is the 427 construct as depicted i,n Figure 14. , 3. A transgenic pig co prised, of the DNA sequences encoding human alpha globin ,and human beta globin operably linked to promoter elements where human hemoglobin is produced in at least some of the red cells of said pig and in which, the, amount pf human globin produced relative t , ptal hemoglobin is at least twenty percent. • < -. 4. A transgenic pig comprised of a DNA sequence comprising the pig adult β- lobin regulatory region as contained in plasmid pGem5/Pig pr(K) , deposited with the American Type Culture Collection and assigned accession number 75371, operably linked to a gene, in which the gene does not encode pig adult β globin, where the gene is expressed in at least some of the red blood cells of said pig. 5. The transgenic pig of claim 4 in which the gene is human β globin. 6. • The transgenic pig-,of claim 4 in which the gene encodes a non-globin- protein. 7. A transgenic pig comprised of a DNA sequence comprising the 13 * region of the pig adult β globin gene, as contained in plasmid pPig3'/3, deposited with the American Type Culture Collection and assigned accession number 75372, operably linked to a gene, in which the gene is not pig adult β globin, where the gene is expressed in at least some of the red blood cells of, aid pig. 8. The transgenic J?ig of claim 7 in which the gene is human β globin. .' ' - . ι 9. The transgenic.pig of claim 7 in which the gene encodes a non-glpbin protein. 10. A purified and isolated nucleic acid comprising: the pig adult β globin regulatory region as comprised in plasmid pGem5/Pig/3pr(K) , as deposited with the American Type Culture Collection and assigned accession number 75371. 11. A purified and isolated nucleic acid comprising: the pig e globin gene as comprised in plasmid pSaf/pige (K) , as deposited with the American Type Culture Collection and assigned accession number 75373. 12. A purified and isolated nucleic acid comprising: the 3' region of the pig adult β globin gene as comprised in plasmid pPig3' 3, as deposited with the American Type Culture Collection and assigned accession number 75372. 13. A transgenic pig comprised of the DNA sequences encoding human alpha globin and human beta globin operably linked to promoter elements where human hemoglobin is produced in at least some of the red cells of said pig and in which the nucleic acid encoding human alpha globin or human beta globin comprises a mutation which increases the level of authentic human/human dimer in the transgenic pig. 14. The transgenic pig of claim 13 wherein the mutation in human alpha hemoglobin is selected from the following group of alpha-chain mutations: a Thr at position 30 instead of Glu; a Tyr at position 36 instead of Phe; a Phe instead of Leu at position 106; a Ser or Cys instead of Val at position 107; and a Cys instead of Ala at position 111. lβ. The transgenic pig of claim 13 wherein the mutation in human beta hemoglobin is selected from the following group of beta-chain mutations: a Leu instead of "Val at position 33; a lie instead of Cys at position 11J * a Val or Leu instead of Ala at position 115; a His .. nstead of Gly at position 119; a Met instead of Pro at position 128; and a Glu instead of Gin at position 131. 16. The transgenic pig of claim 15 wherein the mutation in human beta hemoglobin is a Cys to Val change at position 112. 17. A transgenic pig comprised of the DNA sequences encoding human alpha globin and human beta globin operably linked to promoter elements where human hemoglobin is produced in at least some of the red cells of said pig and in which the nucleic acid construct is the hemoglobin Presbyterian construct as depicted in Figure 1G. 18. A method for purifying human Presbyterian Hemoglobin from a mixture of human hemoglobin, pig hemoglobin, and human/pig hybrid hemoglobin, comprising: (i) collecting red blood cells from a transgenic pig according to claim 17; (ii) releasing the contents of the collected red blood cells to produce a lysate; (iii) applying the lysate of step (ii) to a High Q resin column equilibrated with 20 mM Tris-Cl and 20 mM Glycine at a pH 8.1; ( (iv) eluting the column with a linear salt gradient of 9-16% in buffer containing lOmM Tris-Cl, 20mM Glycine, 250mM NaCl at pH 8.1; and (v) collecting the fractions that contain purified human Presbyterian Hb. 19. A transgenic pig comprised of the DNA sequences encoding human alpha globin and human beta globin operably linked to promoter elements where human hemoglobin is produced in at least some of the red cells of said pig and The transgenic pig of claim 1 in which the nucleic acid construct is the hemoglobin Yoshizuka construct as depicted in Figure IF. 20. A method for purifying human Yoshizuka Hemoglobin from a mixture of human hemoglobin, pig hemoglobin, and human/pig hybrid hemoglobin, comprising: (i) collecting red blood cells from a transgenic pig according to claim 19; (ii) releasing the contents of the collected red blood cells to produce a lysate; (iii) applying the lysate of step (ii) to a High Q resin column equilibrated with lOmM Tris-Cl and 20mM Glycine at a pH 8.7; (iv) eluting the column with a linear salt gradient of 5-30% in buffer containing lOmM Tris-Cl, 20mM Glycine, 250mM NaCl ,at pH 8.7; and (v) collecting the fractions that contain purified human Yoshizuka m?. _ i t- W,

Inventors: HOLTZMAN STEVEN H, KELLER HILARY, KUMAR RAMESH, LOGAN JOHN S, O'DONNELL J KEVIN, PARSONS CYNTHIA T, PILDER STEPHEN H, PINKERT CARL A, SHARMA AJAY, SWANSON MARK E, WHITE STEVEN P, HOLTZMAN, STEVEN, H., KELLER, HILARY, KUMAR, RAMESH, LOGAN, JOHN, S., O'DONNELL, J., KEVIN, PARSONS, CYNTHIA, T., PILDER, STEPHEN, H., PINKERT, CARL, A., SHARMA, AJAY, SWANSON, MARK, E., WHITE, STEVEN, P.
Applicants: DNX CORP, DNX CORPORATION
Application year: 1993
Main classification: A01K67
IPC codes: A01K67, A61K38, A61P7, C07K14, C12N15, C12P21