AU629169B2 - Method of creating new breeds of mammals - Google Patents

Description

AU-Al-17004/88 PCT WORLD [,NTELWGrL. ROftY INTERNATIONAL APPLICATION PUBJLISHED DE? fTI COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 83/ 08026 C12N 1/100, C12Pj 17/34 A (43) International Publication Date: 20 October 1988 (20. 10.88) (21) Internationa! Applicatiou, Number: PCT/AU88/00109 (74) Agent: PHILLIPS ORMONDE FITZPATRICK; (22) International Filing Date: 14 April 1988 (14,04.88) 37ClisSreMlore X30 A) (81) Designated States: AT (European patent), AU, BE (Eu- (31) Priority Applicai'n Numbers: PI11427 ropean patent), CR (European patent), DE (Euro- P1 5326 pean patent), DK, FI, FR (European patent), GB (European patent), IT (European patent), JP, LU (32) Priority Dates: 14 April 1987 (14.04.87) (Europtmn patent), NL (Erpa U 9(uo November 1987 (10,11.87) pean patent), US.

(33)_Priority Country: AL 1 (7)SECTION 34(4)(a) DIRECTION SEE FOLIO) f, mM ig h ng theip NAME DIRECTED 0Aet Ihercep (72N Inventors/Applicants Uor "037T 7Et& W oty r. sii- c-IP bert, Frederick jAU/AU]; 34 Union Street, Bealtlah Park, S.A. 5067 WELLS, juijan, Richard, te AUSTRALIAN [AU/AU]; 1 Torrens Street, College Park, S A, 50t)9I 4 NOV 1988 PATENT OFFICE (54) Title: 1 lITEtV J1,..L (57) Abstract YY-\ CA W\ CaN S A methrnd for rattng new breeds of aitials which comprise. obtaining a recently fertilised ovum, isolating a gene sample of a characterising hormone ho.mologous with the ovum, 1,c) inzroucing t' gene sample into the male nucleous of the ovum prior to fusion with the female nucleous to form s~ngle cell embryo-, and subsequently implanting tho ovum into a suitably prepared female animal, Wherein In a prefrrmd method there is used a plasmid exprebsion vector comprising a plasmid cloning vector including a first cloned sequence of DNA encoding a non-porcine promoter region and a second cloned sequence encoding porcine growhct hormone acti~ity, -d II I I S O 8/08026 PCT/AU88/00109 -1- This invention relates to animal husbandry and in particular to methods of creating new brecds of animals having desired characteristics, such as, increased weight gain, feed efficiency, milk production or disease resistance.

In traditional breeding processes is has been possible to achieve animals with particular desired characteristics. However, many unwanted, as uell as the desired characteristics are often obtained in such processes.

Recently there has been major developments relating to the introduction of exogenous genes into the gene line of animals. In general, this is achieved by the use of both R/DNA technology (which includes isolation and characterisation of a gene) and one-ccU embryo techniques, including egg collection and re-implantation, In summary the overall method involves the following steps: isolation of a pure gene sample (a fragment of

DNA;

collection of a recently fertilised egg; -9 injection of a tiny portion 10 of one cube centimetre) of the gene solution into the male nucleus (from the sperm) before it fuses with the female nucleus to form the one-cell embryo; and implanting the injected egg into a suitably prepared surrogate mother.

After the offspring is born, a small portion of tissue (usually the tail) is taken and DNA is prepared therefrom which can be assessed to establish whether the gene injected at the one-cell stage has been stably incorporated into the animal's chromosomes. If so, it is transgenic.

S |The process described above has been successfully used to accelerate growth and increase size in mice by the introduction of a gene construct incorporating exogenous genes, such as human growth hotone. However, the process has not been successful in attempts to enhance desired characteristics in farm animals.

It is an object of the present ir.vention to overcome, or at least alleviate, one or more of the A4 difficulties and/or deficiencies related to the prior art 0 processes.

-la- According to one aspect of the present invention there is provided a method for preparing new breeds of mammals excluding human boings, said method comprising the steps of: obtaining a recently fertilized mammal ovum; isolating a first DNA sequence encoding a suitable promoter region; inserting the first DNA sequence into a plasmid cloning vector; isolating a second DNA sequence encoding a growth hormone gene homologous with the ovum; inserting the second DNA sequence into the plasmid clc ,.nq vector at a suitable site such that the first DNA sequence can act as a promoter for expression of the second DNA sequence upon transgenesis; introducing the plasmid cloning vector into the male pronucleus by microinjection prior to fusion with the female nucleus to form a single cell embryo; subsequently implanting the ovum into a female animal and allowing the embryo, resulting from intruduction of the plasmid cloning vector into tha ovum, to develop to maturity.

allowing the transgenic embryos to develop to maturity and then testing each individual for successful insertion and expression of the growth hormone.

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j O I ^l Ig i The recently fertilised ovum may be of any suitable animal.

Suitable animals include farm animals such as horses, cows, pigs, sheep, goats, turkeys and also marine animals such as abalone. The fertilised ovum may be obtained by any known method.

S The gene sample of a characterising hormone homologous with the ovum will necessarily be dependent upon the animal egg obtained. For example, if the animal egg obtained is of a pig then the gene sample will comprise characterising pig hormones.

A "suitable promoter region" as used herein the claims and 39 description is a region present in a promoter sequence which is capable of influencing the promoter seque, re to act as a promoter for expression of a DN~A sequence upon trans,- enesis.

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CI IWO &Q,08026 PCT/AU8/00109, 2 Accord-ing -b o t oe-- Zspe -et f-I. ILe- e i I ViLL ivm Lk' there is provided a method for creating new breeds of -'nimals which comprises: obtaining a recently fertilised ovum; isolating a gene sample of a charact ising hormone homologous with the ovum; introducing the gene sample in the male nucleus of the ovum prior to fusion wit the female nucleus to form a single cell embryo, and subsequently implantin the ovum into a suitably prepared female ani The recently fert' ised ovum may be of any suitable animal. Suitable anima include farm animals such as horses, cows, pigs, eep, goats, turkeys and also marine animals auch a ab one. The fertilised ovum may be obtained by any known me od.

The ene sample of a characterising hormone homologou with the ovum will necessarily be dependent upon the ani al egg obtained, For example, if the animal egg obta' ed is of a pig then the gene sample will comprise cccterising pg hormones.- The type of characterising hormones isolated and injected may be any desired characteristic hormones. The type of hormones isolated and injected may be dependent upon the desired characteristics of the off-spring. For example, if the off-spring is a porcine animal and the desired characteristic is accelerated growth, the gene sample may comprise porcine growth hormone. The gene sample may be isolated by any known method.

According to a further aspect of the present S invention there is provided a plasmid expression vector including a first cloned sequence of DNA encoding a non-porcine promoter region ard a second cloned sequence of DNA having porcine growth hormone (PGH) activity.

The plasmid expression vector so formed may include a complete copy o. the porcine growth hormone in the coding region.

Any suitable plasmid expression vector or cloning vector may be used. ror example the plasmid cloning vector pUC19 (Nonander et al, 1983 Gene 101-106) has been found

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The first cloned sequence of DNA may encode a human promoter region. The human promoter region may be the human metallothioneine promoter (hMTIIA). In a preferred form, a modified metallothioneine promoter plasmid may be used.

The first cloned sequence of DNA may form an Eco RI Hind III insertion in the plasmid cloning vector. This insertion may be approximately 810 823bp in length. In a preferred embodiment it is 823bp in length. The first cloned sequence of DNA may also contain elements of the human metallothioneine promoter.

The DNA sequence coding for porcine growth hormone activity may be isolated from a cDNA library formed from messenger or polyadenylated RNA. For example this may be oo" isolated from porcine pituitary tissue.

"The second cloned sequence of DNA ray form an Eco RI Eco RI insertion in the plasmid expression vector. This insertion may be approximately 522 b? in length. The plasmid is a small, high copy number expression vector. The plasmid expansion vector may be plasmid pUC19 or pKT52.

The plasmid according to the present invention may further include a third cloned sequence of DNA. The third cloned sequence may include the 3' end of the porcine growth hormone gene. The third cloned fragment may form an Sma Bam/Sma insertion in the second cloned fragment of DNA.

This insertion may be approximately 1000 bp in length. For example the PGH Gene isolated and characterised for both the coding region and the 3' recion by Vize, P.D. and Wells J.R.E. Gene 55, 339-344 (1951) is suitable.

30 The 3' end fragiient may also be modified. In one embodiment the certain regions identified as repeated sequences are deleted. For example the repeats mi be deleted to leave approximately 200 base-pairs of 3' fragment as opposed to approximately 1000bp in the original sequence. The modifying of the 3' end of the porcine growth hormone DNA sample to remove repeated sequences is an -I~L1 ~I II i" i additional contributing factor to stabilise chromosomal rearrangements of transgenes in the pig genome. This stabilisation may also be achieved by including introns in the porcine gene coding region.

The repeated sequence may cause unfavourable rearrangements in transgenes. This problem is not elcounted with growth hormone genes from other species. Removal of these repeats still showed that positive results could be obtained, this being an unforseen result.

It has been found surprisingly that the inclusion of the 3' end fragment to the coding region may also provide added stability to the read-out sequence (the messenger RNA or mRNA) from the gene.

A particular plasmid of the type described above is that designated pH MPG.4, a sample of which is maintained in the culture collection of the University of Adelaide, Australia. Accordingly in a preferred aspect of the present invention, there is provided the plasmid pHMPG.4. A diagr of the pHMPG.4 construct is provided in Figure 1.

There is also provided a further plasmid wherein the repeated sequences are deleted to provide a plasmid which affords greater stability when incorporated into the DNA.

Accordingly there is provided a plasmid S3 ee

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Si A particular plasmid of the type descri above is that designated pH MPG.4, a sample of w ri~ is maintained in the culture collection of the 'ersity of Adelaide, Australia. Accordin n 'a preferred aspect of the present invention, he is provided the plasmid pHMPG.4. A diagram In a further aspect of the present invention there is provided a method of preparing a plasmid expression vector as described above which includes providing a suitable plasmid cloning vector, a first fragment of DNA containing a non-porcine promoter, and a second fragment of DNA coding for porcine growth hormone; inserting the first fragment of DNA into the plasmid cloning vector at a suitable site; and inserting the second fragment of DNA into the plasmid cloning vector at a suitable site.

The method according to this aspect of the present invention may further include: providing a third cloned fragment of DNA including the 3' end of the porcine growth hormone gene from the chromosomal copy of the porcine growth hormone gene.

This further step may be achieved by cleaving the restrf ited plasmid expression vector at a restriction site for th, porcine growth hormone DNA portion and cloning the third fragment of DNA into the second fragment at a suitable site.

These steps are summarised in Figure It will be understood that the method of preparing a plasmid so described may include the preliminary step of characterising the coding region and when relevant the 3' non-codlng region of porcine growth hormone. This characterisation of porcine growth hormone is described below. The neucleotide sequence of the PGH 4 geen-i± gene is illustrated in Figure 3.

The plasmid or gene sample, for example the porcine growth sample Hind III/PvuI segment derived from the plasmid shown in Figure 1, may be introduced into the male pronucleus of an ovum of a porcine animal by any suitable method. The 'WO 8/08026 PCT/AU88/00109 5 gene sample may be injected into the male pronucleus of the ovum.

In a preferred aspect of the present invention, there is provided a method of preparing a transgenic animal including: providing a recently fertilized ovum from a female of the animal; preparing a plasmid expression vector containing the first and second cloned fragments of the promoter and cloning regions of the gene to be expressed, and injecting the plasmid into the male pronucleus prior to fusion with the female nucleus to form a single cell embryo.

A major problem encountered with the injection of the gene construct into the male pronucleus is visualisation of the pronuclei or nuclei in the ova. Nuclear structures of such animals as rabbits may be readily seen. However, pronuclei and nuclei in such animals as sheep and pigs are difficult to locate.

Pronuclei and nuclei of sheep ova may be seen by fluorescent microscopy using DNA specific fluorochromosomes or by interference contrast (IC) microscopy. The combination of stain and ultraviolet light may be damaging to the ovum, Therefore interference contrast microscopy is preferred for microinjection of sheep ova. Fluorescent analysis has indicated that interference contrast microscopy is an effective method for pronuclear localisation in approximately of fertilised sheep ova. A similar process may be used to visualise pronculei and nuclei of goat ova.

Pig ova are opaque and no nuclear structures can be seen even with interference contrast microscopy. However, the pronuclei or nuclei of pig ova may be visible if natural pig ova is centrifuged at 15000 g for 3 min. A similar process may also be used to visualise pronuclei and nuclei of cow ova. However, centrifugation does not help visualisation of pronuclei of sheep ova.

The injection of the pronucleus may be carried out under magnification and use of standard microinjection apparatus. The ova may be held by a blunt holding pipette and the zona pellucida, plasma membrance and pronuclear I I I I I ft WO 88/08026 PCT/AU88/Q0109 6 envelopL may be penetrated by an injection pipette. The blunt holding pipette may have a diameter of approximately pm. The injection pipette may have a diameter of approximately 1.5 pm.

The amount of gene construct injected will necessarily be dependent upon the size of the pronucleus and the size of the injection pipette. For example, a few hundred copies of a 2.6 kilobase (kb) linea fragment containing the desired gene characteristic hormone may be injected.

The egg may be subsequently implanted by any suitable method into any suitably prepared surrogate mother.

This may be achieved by any known method.

To further illustrate the invention the following non-limiting example is provided: EXAMPLE 1 PGH cDNA sequences A porcine pituitary cDNA library of approximately 4000 individual recombinants constructed using the plasmid vector pUC19 (Norrander et al., Gene, 26, 101-106 1983) allowed the isolation of PGH cDNA clones using a synthetic DNA probe. One of the cDNA clones isolated, pPG.3, which was found to contain an insert of the e'pected full length, was completely sequenced.

The open reading frame of the pPG.3 cDNA insert was found to code for a 216 amino acid pre-hormone, identical in amino acid sequence to the partial length PGH cDNA clone of Seeburg et al. DNA, 2, 37-45 (1983). This clone provided for the first time the complete sequence of the region encoding 30 the PGH signal sequence and 41 basis of the 5' untranslated region.

The nucleotide sequence within the coding region is very highly conserved, with only a single base difference from the sequence of Seeburg et al (1983).

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Nucleotide sequence UL the PGI genomic ene The complete 2231 bp sequence is illustrated. The open reading frame which enc\odes pre-PGH is indicated, as are basis which differ in sequence from the previously studied PGH cDNA sequence,pPG.3. Base changes which result j 1 i i s t" i ON- I WO 88/08026 PCT/A IU88/109 18 WO 88/08026 PCT/AU88/00109 7 in animo acid substitutions are marked with an asterisk. The single base in the 3' untranslated region which differs from pPG.3 is also indicated below the sequence. The location of the cap site was inferred from the position of the rat and human GH gene cap sites (Page 35 al., 1981; DeNoto et al., 1981).

Putative promoter and polyadenylation sequence, such as the TATA, AATAAA and GT-rich sequences are underlined, and the position at which the poly A tail is added is indicated with an arrow. The variant GC donor splice site is located in the first intron, around base +72.

The Smal restriction site utilized for subcloning in latter Chapters is indicated.

Southern analysis using a PGH cDNA hybridization probe When the cDNA insert of pPG.3 was used to probe Southern blots of porcine genomic DNA digested with three different restriction enzymes, a single strongly hybridizing band was detected in each track. Enzymes BamHI and EcoRI also produced a second, fainter band, indicating that if the gene was in fact present as a single copy, the genomic gene must contain internal sites for both of these enzymes, somewhere near the end of the region homologous to the cDNA (as was later discovered to be correct). The third enzyme used, Hind III, produced only a single clear band. Taken together these data indicated that only a single copy of the GH gene exists in the porcine genome (per hapoid chromosome comlement), a situation analogous to the bovine and rat genomes, but very different to that of humans.

Isolation and analysis of the genomic PGH gene The PGH cDNA clone pPG.3 was used to screen a Sporcine cosmid library and isolate a clone containing PGH gene sequences Southern analysis of the cosmid clone identified major BamHI and EQoRI hybridizing bands where were equal in size to those detected in genomic Southerns utilizing the same hybridization probe, Nucleotide sequencing of the gene contained within the cosmid revealed that the entire coding region was present (648 bp), along with 178 bp of the promoter region, 61 bp of 5' untranslated sequence, four introns of 242, 210, 197 and 278 bp a- WO 88/08026 PCT/AU88/Q0109'.

respectively, and 414 bp of 3' non-coding sequence (Figure The gene contains four base alterations within the coding region relative to the prev 4 -s~ly sequenced PGH cDNA clone, pPG.3. The base substitut' hich are illustrated in Figure 3, result in the alteracioni of two amino acid residues within the signal peptide. The remaining two differences are silent substitutions. There is also one base change in the 3' untranslated region relative to the pPG.3 cDNA sequence (Figure 3).

The sequence comparison illustrated in Figure 4 indicates that the promoters of each of the studied sequences share a high degree of homology. Figure 3 indicates that the untranslated regions of the GH and HPL genes are als;o conserved to a surprlsing degree.

Analysis of t-he 3' sequences of the PGH gene allowed the identification Of sequences which have been shown to be important in the polyadenylation process.

The comparison of the 3' sequences of the PG3H gene to all of the available GH and HPL squences. has revealed that these are also conserved to a surprising extent, between both GH and HPL genes.

Ficluxge 4 Cpmparison of-GH- and HPL gene Promoter untranslated seonuences Porcine bovine human and rat (tR) GH genes and tho Yhuman placental lactogen (HPL4) rte we~re aligned to determine the extent of sequence hcorology (~.,.a).Asterisks indicate homology betweenan t sequences, The PGH sequence is numberzed with respect to tho distance from the cap site This example desc-ibes the cloning of the por. ilne GH cDrNA insert contained in! pPG.3 into a bacterial Fexpression vector.

EpRes"Ision of PGH in E t Qo1 The expression vector chosen Eor the production of PGH in R, _I 06'110 was pKT52, This plasmid, kindly provided by a, Shine (California Biotochnolocgy)i is a smal.l, high Copy number expression vector which coatains a powerfu, requlatable zx-, promoter (Brovius et al., 1 and the U V :II" .WO 8s/08026 PCT/AU88/00109 9 strong E.coli 5S transcription terminators (Brosius et al, 1981). The trc prc.noter is a fusion promoter containing the consensus -35 region of the E.coli =trp promoter moined to the consensus -10 region of the E.coli lacUV5 promoter (do Boer et al., 1983a). The lacUV5 sequences of this promoter contain a lac operator site, which results in transcription from this promoter being repressed in lacIq strains.

Transcription from this promoter in la_. Iq strains can be stimulated by growing cells in the presence of IPTG (de Boer et al., 1983a). A restriction map and the sequence of the RBS/ cloning region of pKT52 is shown in Figure Figure Cloning PGH cDNA into expression vector pKT52 The organization of the E.coli expression vector pKT52 is illustrated. An 800 bp PstI fragment was isolated from M13 RF containing the EcQRI insert of pPG.3. This fragment contained the entire pre-PGH coding region, minus the first two amino acids, plus 33 bp of mpl9 polylinker DNA. When this fragment was inserted into the PstI site of pzT52, the full pre-PGH sequence is regenerated The E9gRI insert of pKT52 was then isolated and cloned into M13 mpl9 to facilitate mutagenesis of the pKT52/PGH cDNA junction point The position of the trc promoter and the rRNA transcription terminators (rrnTl and rcnT2) are indicated.

Two E.coli strains were used for analysing expression levels n the work described in this Example, strains MC1061 and JM101. Expression from the trc promoter is constitutive inMC1061 cells, and repressed in the lac Iq strain, JM101. The repression of transcription from the trc promoter in JM101 cells was released by growing cells in the presence of 1 mM IPTG.

Plasmid pGHX.1 A construct designed to express methionyl-PGH (m-PGH) was constructed using oligonucleotide directed rutagenesis to delete the DNA coding for amino acids 2-26 of the pre-hormone. This deletion joined the TTc codon of the first amino acid of the mature PGH molecule directly to the ATG initiator codon.

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WO 88/08026 PCT/AU88/00109 10 The EcoRI insert of pKTGH, which contains the entire PGH cDNA fused to the trc promoter sequences, was isolated and cloned into M13 mpl9. Single-stranded DNA isolated from this 'phage was then used in mutagenesis reactions. To remove the 75 bases coding for amino acids 2-26 of the pre-hormone a 30 base long oligonucleotide, bases complementary to 15 4 ba s=s either side of the required deletion was used in a mutagenesis reaction. Following annealing and extension the mutagenesisreaction was transformed into JM101. The resulting plaques were then screened to detect the required deletion by plaque hybridization of/dljGiFt ,e lifts made from the transformation plates. Of the 150 plaques screened 35% were found to hybridize in duplicate to the GH.30 oligonucleotide probe.

Single-stranded DNA wps isolated from a number of positive plaques and sequenced to confirm the accuracy of the deletion using the PGH-specific oligonucleotide, GH.25, as prirrer.

All of the positive plaques contained DNA which had the correct deletion. Replicative form (RF) DNA was isolated from this deleted 'phage (mpGHX.,) and the EcoRI insert purified and subcloned into the larger EcoRI fragment of pKTGH to create plasmid pGHX.l. The nucleotide sequence of the 5' end of this, and the o her expression plasmids described are illustrated in Figure 6, Figure 6 Nucleotide sequence of 5' regions of PGH expression plasmids The nucleotide sequence of the final 24 basis of the mRNA leader and the 5' end of the coding region of each of the expression plasmids generated in Chapter 3 are illustrated. Each of the sequences were determined by priming sequencing reactions wi' the PGH specific oligonucleotide, GH.25 (Figure 5; Each of the plasmids with the exception of pKTGH (pre-PGII) and pGHXF (PGH fusion protein) encode methionyl-pGH (m-PGH). Bases of the m-PGH expression plasmids which differ from the pGHX.1 sequence have been underlined.

PGHXS (sgacer) plasmids A 38 base oligonucleotide with one redudnancy,

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7 f7xtt CGH.38, was designed to alter the RBS from AGGAAA to AGGAGG ',WO "/08026 PCT/AU88/00109 11 and the spacer from CAGACC to either TAATAT or TAAAAT.

Single-stranded DNA containing the small EcoRI fragment of pGHX,1 was mutagenized and positive plaques selected and sequenced. A total of 30% of the plaques hybridized to the GH.38 probe, but of these only 20% contained either the GGTAATAT or GGTAAAAT RBS/spacer sequences. The remaining positives contained duplicate insertions which may have arisen due to the incorrect hybridization of the GH.38 oligonucleotide during the mutagenesis reaction. RF DNA was prepared from plaques containing correct versions of both the required alterations and then cloned back into pKTGH. The nucleotide sequence of the RBS/spacer region of the resulting plasmids, pGHXS.4 and pGHXS.9 are illustrated in Figure 4.

Extracts prepared from both MC1061 and induced JM101 cells containing either of these two plasmids were found to contain an additional prominent band, which had a molecular weight of 22 K, the expected size of m-PGH, Laser densitometry of SDS/PAGE gels indicated that both plasmids produced identical levels of m-PGH, which ranged in the two hosts from 15 to of total cellular protein.

Figure 7 illustrates the level of m-PGH produced from pGHXS.4 in both uninduced and induced JM101 cells. This Figure also illustrates the similarity in molecular weight of the E.coli produced protei,i versus pituitary derived PGH.

The m-PGH migrates at a slightly higher molecular weight than expected (anproximately 200 daltons), probably due to the extremely crude nature of the protein extracts applied to the gel. This phenomenon has been previously observed in crude bacterial extracts containing human GH (Hsiung et al., 1986).

Fiqure 7 Production of m-PGH by plasmid pGHXS.4 Protein extracts from uninduced (-IPTG) and induced (+IPTG) JM101 cells containing expression plasmid pGHXS.4 were subjected to SDS/PAGE, along with molecular weight markets and purified, pituitary derived PGH (kindly provided by R, Seamark). A prominent band of the expected molecular weight of PGH (22,000 daltons) is produced only in IPTG induced cells (3.2.2.iii).

L 12 The construction of a human MT-IIA promoter/PGH fusion gene The promoter chosen for these studies was the human metallothioneine II-A promoter (Karin, M. and Richards, R.I.

:ature 299, 797-802 (1982) which was kindly provided by R.

Richards.

Ihe hMT-IIA promoter was available as an 823 bp fragment with promoter sequences extending from -763 to cloned in the M13 vector mp8 Robbins pers. comm.). The double digestion of RF 'phage containing this promoter with HindIII and EcoRI releases the promoter sequences fused to bp of vector polylinker sequence, 'Znis 823 bp fragment was purified and subcloned into HindIII/EcoRI digested pUC19 to generate plasmid pUCMT.

e The sequences encoding PGH were isolated from the PGH as cDNA clone pPG.3 as an 814 bp EcoRI fragment. This, w cloned downstream of the hMT-IIA promoter, by restricting pUCMT with EcoRI, followed by ligation to the puriied pPG.3 insert. Restriction analysis of plasmid DNA prepared from the resulting transformants (6.3.4.ii) identified a plasmid which contained the PGH cDiA inserted in the correct orientation, which was named pUCMTGH.4 (Figure The nucleotide sequence of the junction point between these two fragments was determined by the directional subcloning of a restr 4 ,ction fragment spanning this region into M13 mpl8.

The sequence data derived from this clone indicated that the expected sequence had been generated, and had contained the hMT-IIA promoter sequences and transcription start site (down to position +60) joined to the PGH 5' untranslated region (from +21 onwards) by 9 bp of polylinker/synthetic 30 linker DNA (Figure 8).

It was decided to subclone the 1 kb SmaI/BamHI fragment from plasmid pGHB.3 (2 kb BamHI subclone of cPGH.1) whiah contains the fifth exon downstream of the Sma I site plus approximately 800 bp of 3' non-translated sequence from the pgh gene into the unique gm_.I site of pUCMTGH.4.

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-128- The 1 kb SmaI/BamHI fragment of pGHB.3. was purified (6.3.2.ii) and the BamHI generated overhang repaired with the Klenow fragment of E.coli DNA polymerase I. This blunt-ended fragment was then subcloned into SmaI digested pUCMTGH.4. The examination of plasmid DNA isolated from a number of the resulting transformants revealed that most were equal in size to pUCMTGH.4. The transformants were therefore screened for the presence of the cPGH.1 sequences by filter hybridization using a 500 bp BamHI/PtI restriction fragment from the far 3' end of pGHB.3 as the hybridization probe. Restriction analysis of plasmid DNA prepared from the resulting positives indicated that one contained the inserted fragment in the correct orientation. This plasmid was named pHMPH.4 (HM, human metallothionein; PG, pricin growth hormone). The organization of this plasmid is illustrated in Figure 1.

To produce the modified plasmid pHMPGA.5, the 1kb SmaI/Ba_4AI fragment from plasmid pGHB.3 was purified and digested with Bal31 prior to subcloning into SmaI digested pUC MTGH.4.

*too .25 too: t. g 1**35 The transformants were screened for the presence of the cPGH.1 sequences by filter hybridization using a 500 bp BamHI/PstI restriction fragment from the far 3' end of pGHB.3 as the hybridization probe. Analysis of the positives yielded a plasmid in the correct orientation which was named pHMPGA.5. This plasmid was used to create transgenic animals.

b E( I LI-CIIPI VWO 88/08026 PCT/AU88/00109 13 "mCL~b.c C~ ~hCI1~~ C~~lim~.~ -==wje~F er_--eEr 11 a, .1 inS:J;,t to pUCMTCH.4. The transformants were therefore screene for the presence of the cPGH.1 sequences by filter fb idization using a 500 bp BamI/PstI restriction fra .n c from the far 3' end of pGHB.3 as the hybridization obe. Restriction analysis of plasmid DNA pre rd from the resulting positives indicated that one co ned the inserted fragment in the correct orient on. This plasmid was named pHMPH.4 (HM, human me othionein; PG, porcine growth hormone). The or a i a Rp.and== 3 Fiqure 2 Construction of eukaryotic expression plasmid pHMPG.4 A flow chart illustrating the construction of pHMPG.4 is illustrated. An approximate 800 bp HindIII/EcoRI fragment containing the hMT-IIA promoter was cloned into HindIII/EcoRi digested, dephosphorylated pUC19 to create plasmid pUCMT. This plasmid was then restricted with EcoRI, dephosphorylated, and ligated to the EcoRI insert of the PGH cDNA clone, pPG.3, to generate plasmid pUCMTGH.4. This plasmid was restricted with SmaI, dephosphorylated, and ligated to the blunt-ended 1 kb SmaI/BamHI insert of cosmid subclone pGHB.3, which contains most of the last exon of the PGH genomic ge.-e and approximately 700 bp of PGH 3' PHMP tG.

non-coding sequence to create 4 pM4 4. All constructs were checked by DNA sequence analysis..

Figure 8 Nucleotide secuence of the hMT-IIA/PGH junction point in pUCMTGH.4 A ScaI/PstI fragment which covers the junction region between hMT-IIA and PGH cDNA sequences was isolated from pUCM'"GH.4 and cloned into SmaI/PstI digested mpl8 for sequence analysis. The sequence data from this clone indicated that the correct fusion had been generated, with the hMT-IIA promoter, cap site (there are two cap sites, both indicated as and 5' untranslated sequences (extending down to linked to the PGH cDNA sequences, which extend downstream from base +21, by 9 bp of synthetic linker sequence Vtc

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WO 88/08026 PCT/AU88/0109 14 Figure 1 Expression vector, pHMPG.4 The restriction map and organization of expression vector pHMPG.4 is illustrated. The hMT-IIA promoter sequences are fused to a hybrid gene containing PGH cDNA sequences extending from +21 down to the unique SmaI site, joined to PGH genomic gene sequences downstream from this point. The 2.7 kb HindIII/PvuI fragment containing all of the promoter and PGH sequences, plus 120 bp of the pUC19 lacz gene joined to the 3' end, was purified from low melting temperature agarose, and used for generating transgenic animals.

Production of transcenic pigs pH P The HindIII/PvuI fragment of4pHMPRff was used to produce transgenic pigs. Single cell in vivo fertilized pig embryos were collected from superovulated large white sows.

These were prepared and injected with approximately 600 coe of te p ^.s copies of the p4 insert, Approximately 30 injected embryos were surgically transferred into the oviducts of each of a number of synchronized recipient sows. Four of these sows farrowed small litters (4-5 per litter), producing a total of 17 piglets.

Analysis of transqenic pigs i) Dot-blots The piglets were tested for the presence of the foreign gene by dot-blot hybridization of DNA isolated from tail tissue. Both normal pig DNA and human genomic DNA were included on these dot-blots to act as negative and positive controls respectively. Following the hybridization of these dot-blots to the HindIII/Aval fragment of the hMT-IIA promoter a number of positive signals were evident. Four of the pigs showed strong hybridization equivalent to greater than one copy per cell and a further two showed waak hybridization, slightly above background, and equivalent to less than one copy per cell (Figure 9).

ii) Slot-blots e A tH.G- The number of copies per cell of the pIPrPG.'insert present in each of the tranigenic pigs was determined by slot-blot analysis. Five pg samples of tail DNA from each of WO 88/08026 PCT/AU88/00109 15 the transgenic animals was filtered onto a slot-blot along with human and pig positive and negaitve controls. A range of aiTounts of pHMPGA,4 plasmid DNA (which also contained 5 pg of control pig genomic DNA) corresponding to genomic copy numbers equivalent to between one and fourty copies per cell were also includad. This slot-blot was hybridized to the nick-translated IiindIII/AvaI fragment of the hMT-IIA promoter and washed at high stringency. The intensity of hybridization of the transgenic animals was compared to the plasmid standard s by laser densitometry, and found to range from approximately 0.5 copies per cell in animals #375 and #739 to 15 copies per cell in animal #295 (Table 1; Figure 9).

iii) Southern Analysis The organization of the foreign sequences within the transgenic pigs was studied by Southern blotting. There are no BamHI sites within the pHMPGA.4/insert. The digestion of the genomic DNA of the transgenic animals with this enzyme should therefore produce bands on genomic Southerns, the length of which are governed by the distance of the nearest BamHI sites to the site of integration, the number of integration sites, and the number of integrated copies of the pHMPG4i insert.

Figure 9 Analysis of potentially transgenic pigs by dot-blot 10, 5, and 1 pg samples of DNA isolated from the tails of pigs which had developed from eggs microinjected with the insert of pHMPGA, were denatured and applied to a membrane. pHMPG4,jand human (HUM) ~enomic DNA positive controls and pig (PIG) genomic DWA negative controls were included on each dot-blot array. The plasmid positive controls are in row 1, blot A. In this row the numbers in brackets refer to the number of copies per cell each plasmid dot is equivalent to. Samples in cows A4 to All and B1 to B9 contain the test pig samples. The nick-translated hMT-IIA promoter HinIIl/Aval insert ws used as the hybridization probe. A key indicating the identity of the sample on each dot is shown below, I-Ii-- WO 88/08026 PCT/AU88/00109, 16 1 2 3 4 5 6 7 8 9 10 11

A.

B.

C.

10ig (2) 5pg (1) lpg (0.5) HUM PIG 177 178 179 to ii SI I Io 180 295 296 297 298 of ii It Ii I" IF I II 1 2 3 4 5 6 7 8 9 10 11

A.

B.

C.

10ug 373 5pg (1) 1)ig (0.5) 374

I

"1 375 376 735 736 i i is t 737 It 738 739 PIG HUM I to II o I II II II Figure Slot-blot analysis of transgenic pigs Samples (5 pg) of transgenic pig DNA and pig (PIG) and human (HU1) negative and positive controls were denatured and applied to a membrane along with a number of samples

S

containing various amounts of pHMPGA. plasmid DNA combined with 5 pg of pig control DNA. The amounts of pHMPGA.4jDNA applied correspond to gene copy numbers of between 1 and gene copies per cell (shown in brackets below). The probe used was the nick-translated hMT-IIA promoter HindII/Aval insert. A key to the samples on the blot is given below.

Quantitation of the intensity of hybridization of each of the samples on this membrane was performed using a laser densitometer. The results of this analysis are presented in Table below.

A B C 1.

2.

177 375 180 736

PIG

295 739

HUM

6.

7.

500 (40) 375 (30) 250 (20) 125 (10) 100 (8) 75 (6) 50 (4) 25 (2) 12.5 (1) WO 88/08026 PCT/AU88/00109 -17 Table 1 Transqenic pigs: gene copy number, growth, and serum PGH concentration The number of copies (per cell) of the foreign gene present in each of the transgenic pigs estimated by slot-blot analysis, the daily weight gain (between days 50 and 120) and the serum PGH concentration of each of the transgenic pigs and their non-transgenic littermates are illustrated. The female control values are the mean of three animals, and the male control values are the mean of two animals.

Pig No. sex copy no./ daily weight serum PGH cell gain (qm) conc. (nq/ml) 177 F 3 765 10.4 295 F 15 953 27.8 739 F 0.5 686 6.9 controls F 806 10.6 180 M 6 851 15.3 37; M 0.5 487 6.3 736 M 6 857 11.1 controls M 670 15.3 Growtu rate of transqenic pigs The growth performance of each of the transgenic pigs is plitted against the growth rate of non-transgenic littermates. The dotted lines represent the average growth rate of sex-matched non-transgenic littermates, the dashed line represents the average of non-transgenic littermates of the opposite sex, and solid lines represent transgenic animals.

Example 2 Spl binding site minus hMT-IIA/PGH plasmid, piiHGPG.3 It has been reported that a consensus Spl binding site sequence, GCGG (Kadonaga et al., Trends Biochem, Sci.

11, 20-23, 1 96) located adjacent to the basal lev-l sequence would generate a promoter with the desired para ters. The nucleotide sequence of an altered hMT-IIA At AGU"- .the Sp 1 binding, site rlaaL=wbAf -1-i Each transgenic animal is unique and the level of expression f-om incorporated transgenes is highly variable from one to another such variation is common and it represent, problem. The transgenic animals are bred from an animal where insertion and expression have occured as desired as in pig. No. 295.

Figure 11 Growth rate of transcenic piqs The growth performance of each of the transgenic pigs is plitted against the growth rate of non-transgenic littermates. The dotted lines represent the average growth rate of sex-matched non-transgenic littermates, the dashed line represents the average of non-transgenic littermates of the opposite sex, and solid lines represent transgenic animals.

Example 2 Spl binding site minus hMT-IIA/PGH plasmid, pHMGPG.3 It has been reported that a consensus Spl binding site sequence, GGGCGG Kadonoga et al., Trends Biochem. Sci.

11, 20-23, 1986) located adjacent to the basal level sequence, would generate a promoter with the desired parameters. The nucleotide sequence of an altered hMT-IIA promoter which has the Spl binding site replaced with a PstI e*e 9. *r a 4O r a.rr

I

orI WO 88/08026 PCT/A U88/00109 18 linker, and possesses the required transcriptional characteristics, was kindly made available by M. Karin. This alteration was recreated in the hMT-IIA/ mp8 clone by oligonucleotide directed mutagenesis.

A 46 base long oligonucleotide, MT.46 was designed to replace a 15 bp region, surrounding and including the Spl binding site, with a 17 base long PstI linker sequence.

Following mutagenesis of hMT-IIA/ mp8 single-stranded DNA with MT.46 and transformation into JM101 plaques containing sequ en r-e the correctsqttene substitution were selected by plaque hybridization and nucleotide sequencing. RF DNA was isolated from one of the plaques containing the correct mutation and the =tII/EcoRI insert purified and used in a triple R ligation with the EcoRI insert of pHMPG.4 and HindIII/ekeR 4digested pUC19 DNA. One of the resulting plasmids from this ligation contained the correct restriction pattern, and was named pHMGPG.3 (GC sequence minus).

The insert of this plasmid is currently being introduced into transgenic mice.

It is also envisaged that tne process of the present invention mry be used to produce synthetic promoter constructs containing various combinations of metal-responsive Clements corresponding to sequences found in mouse, sheep or human metallothioneine promoters, with or 25 without other promoter elements. For example it may be used to regulate Fe levels.

Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as 30 outlined herein.

i I i'

Claims (20)

1. A method for prepariing4 >aLeai mammals excluding human beings, said method comprising the steps of: obtaining a recently fertilized mammal ovum; isolating a first DNA seqence encoding a suitable promoter region; inserting the first DNA sequence into a plasmid cloning vector; isolating a second DNA sequence encoding a growth hormone gene homologous with the ovum; inserting the second DNA sequence into the plasmid cloning vector at a suitable site such that the first DNA sequence can act as a promoter for expression of the second DNA sequence upon trans-',,,esis; introducing the plasmid cloning vector into the male pronucleus by microinjection prior to fusion with the female nucleus to ,,orm a single cell embryo; subsequently impl;.nting the ovum into a female animal and allowing the embryo, resulting from introduction of the plasmid cloning vector into the ovum, to sees* develop to maturity. allowing the transgenic embryos to develop to maturity and then testing each individual for e .25 successful insertion and expression of the growth hormone.

2. A method according to claim 1 wherein the growth hormone gene from the same nucleus as the ovum is a porcine growth hormone. 30

3. A method according to claim 2 wherein the female animal is a pig. o

4. A plasmid expression vector comprising a plasmid cloning vector including: a first cloned sequence of DNA encoding a non-porcine promoter region; and a second cloned sequence of DNA encoding homologous porcine growth hormone activity.

A plasmid expression vector according to claim 4 wherein the plasmid cloning vector comprises pUC19. -u UP

6. A plasmid expression vector according to claim 4 wherein the plasmid cloning vector comprises pKT52.

7. A plasmid expression vector according to claim 4 wherein the firt cloned sequence of DNA encodes the human metallothioneine IIA gene promoter.

8. A plasmid expression vector according to claim 4 wherein the first cloned sequence of DNA forms an EcoRI-HindIII insertion in the plasmid cloning vector and is approximately 810-823 bp in length,

9. A plasmid expression vector according to claim 4 wherein the second cloned sequence forms an EcoRI-EcoRI insertion in the plasmid cloning 'ector and is of approximately 522 bp in length.

10. A plasmid expression vector according to claim 4 which includes a third cloned sequence of DNA inserted in the second cloned sequence.

11. A plasmid expression vector according to claim wherein the third cloned sequence comprises the 3' 3nd of the porcine growth hormone gene.

12. A plasmid expresion vector according to claim 0#* t wherein the third cloned sequence forms an Sma:Bam/Sma insertion in the second cloned sequence o2 approximately 1000 bp in length.

13. A plasmid expression vecto' according to claim 12 wherein the third cloned sequence is modified by deleting regions identified as repeating sequiences. S:

14. A plasmid expression vector according to claim which is pHMPG.4. 'iOP.

15. A plasmid expression vector according to claim 13 which is

16. A method according to claim 1 wherein the plasmid expression vector is pHMPG.4.

17. A method according to claim 1 wherein the plasmid expression vector is PHILLIPS OR FITZPATRI Attorneys fo:r BRESATgC-- ITED S/OB m -21-

18. A method of preparing a transgenic mammal excluding humans including: providing a recently fertilized ovum from a female of the animal; introducing a plasmid expression vector according to claim 4 into the male pronucleus by microinjection prior to fusion with the female nucleus to form a single cell embryo; subsequently implanting the ovum into a female animal and allowing the embryo, resulting from introduction of the plasmid cloning vector into the ovum, to develop to maturity. allowing the transgenic embryos to develop to maturity and then testing each individual for successful insertion ani, expression of the growth hormone.

19. A method according to claim 1 substantially as hereinbefore described with reference to example 1. A plasmid expressio,. vector according to claim 4 2. substantially as hereinbefore described with reference to example 1. Dated:

20 July 1992 PHILLIPS ORMONDE FITZPATRICK Attorneys for: BRESATEC LIMITED A 'T195j 39 CA 79 i' Xt NOB 0 0 00 0% [yOCIE GROWTH HORMONE EXPRESSION VECTOR, pHMPG.4 Human MT promoter 823bp PGH cDNA 522 bp -Stna PUC19 26239 bp PGH cosmici -1000 bp FkI1 PGH cDNA 299 bp .IWO 98/08026 W 88/8026PCT/ AU8.8/0O 109 2/11. h MT]1A 800bp EE PGH cDNA SO0bp FsMa Sma Scm (blunted) PGH co-smid fragment lC0C0bp FIG 2 Sam/sma WO $08026P~/AU88/0O1O9 3/11 -150 -100 ,AAAATAGGTGGC-OOCAGAGGOkAAAAAGAOCCAOOOG TATMAAA ACGGCCCA ACOOGACCAATTCC A GAATCCCACCACCCAGCTCCCCAGACCACTC -50 +1 met ala ala g AGcGACCTGTGOAGCC0GCTCTG ATG OCT GCAG CGCAAGTOCCCCTAAATCCCAOTCC7TCTTCTTCTGAACOOTGACCGTCCOGCCATOCAG +50 +100 AT~T~GACACr(CTTG CAAGGGAGAKTTCCTGkAGGC A AGC~GGGGGGGA +150 +200 ly pro org thr oer ala lea leu ala MOOCGACArGGGCTGGTOOAGCCAOOCTCTGTCTCOOOATCCCTCTCTCACCCOCCTCCTCTCTCTAG GC CCT COG ACC 7CC GCG CTC CTG CT +250 +300 T* phe ala leu leu eye leu pro trp thr org glu vol gly ala h pro ala met pro let-, oar oer lea phe ala eon ala TTc GCC CTO CTC TOO CTO CCC TOO ACT COO GAG CTG COC C IR CCA CCC ATO CCC T7C TCC AOC CTA rrr GCC &AC GCC +350 A' A 400 val lea org 4la gIn his leu his gin Lau ala ala asp thr tyr lye glu phe OTG OTCOG 00 CC CAG CAC 070 CAC CAA 070 OCT OCT OAC ACC TAO AAO GAG TTT CTkACCTCCCCA~oOAOGCTGCGCGCGTOO +450 C +500 TCGGAAGOGTAATCGTC' TCCGCCTAGTGGCAGO.AATAGGCTG CTATAGGCCCAACATCAGATC=TGAGTGTAAACTGMCCCG +550 +600 glu org ala tyr ile pro giu gly gin ATTCCCAAOrAAAAGOALGCAAOOACAACCGOGCCCCAOTGTAGACcTGGATOGc7TcTCCCCTCCCA~G GAO C00 CCC TAO A'VC CCG GAG OA CAG +650 P700 arg tyr der I,!a gin &I&al gnala la l ph. eye ph. oer gjlu thr 4le pro ala pro thr gly lym aop glu ala gin, AGO TAG TC.U ATC CAO MAC 000 CAG OCT CCC TO TOO TO TCO GAG ACC ATC CCG 0CC CCC ACG 000 AAO GAC GAG CCC CAC +750 gin or& ser CAG AGA TOG GTGA0GOr.CCACCTCCCACCTOCCrACCOGOOAGCACOOOCCTCCCTCrTCCTMGAACGCTCCCCCATCTCTCATCATCAGrGCCIT.CCGCOOC +800 +850 tiop CrCTCOGCAGCTGOO0O7GACGTOCAACOGOTGTA0OGACG=CCACGGCGCAOCCGCCCCCCCATCACCATCGCCCCAG GAO +900 +950 1000 vol gj.u Lou lou org phe der Lau Lau Lou ile gin mar trp Lou gly pro vol gin pho loU der org Val phe thr sort 070 QA G. 70 07000 TTC TOO CTG CTO 070 ATC CAG TOO TOG OTC 000 CCC GTG CAG T0 070 AGO AGO GTC TTC ACC MAC 1050 oer Lou Vol ph. gly thr ear asp org Val tyr gitA lye lou Lysasp lou glu glu gly ii. gin ala lati met arg AGC ,TG 070 TT 000 ACC TCA GAC COO OTC TAO GAO MG 070 AAO GAO CTO GAG GAG 000 ATC CAC GOC CTC ATO COO OT +1100 +1150 +1200 1250 0CCrGTAGCAGCATCTCACOCAGAA07TC7rrCATrCCc0rOCACC07?IGCrrTCTTCAAQCATAGGGOACGTGAAAC +1300 1350 Smna I glu la glu asp gly Bar pro org ala gly gin CGAOOOACAGAACC CTGCCAAGACCOCCTCTGTCTCTCTCTCCCrITTOAG GAG 0C% GAG OAT 000 AGO COO COO CU GGA CAC +1400 A ile iou lya gin thr tyr asp l.ya ph. asp thr son Lou org mar asp asp &Ia Lou Lau lyes on tyr gly lea leu oar ATO 070 MOG ChA ACC TAG GAC AAA TTT GAO ACA, AC TTO COO ACT OAT GAO 000 070 CTT AAO MO TAO 000CT CTC 7 CC +1500 +1550 eye ph. lye lye asp Lou him lye als glu thr tyr lea arg val, not lye eye arg org ph. Val glu %or der eye ala TGC TTC 04AAC GAC CTG CAC AA OCT AGACA TAC CCGO GTC ATG AATOT CCC TC TG GAGACC OTGTOCC 1600 phe 'TOC TAG TTGCTGOCATCTC7TTGCCCCTCCCCAokTAOCTCOO07GACCCTCAA GCCACCCCAATCOGCTOTCCTICCT AATAA ACAGOTTOC +1650 T+1700- ATOOT tATTOTCMOACTA, GOTCACT CT000ATGAGGGAOOCAGAOrGCAAGOGTrGCOTGOA.ACACCTGCAOOCATC=OGOOT0C S+1 75,1 +1800 07CGACCAGACA AAT0ATGGACCCr.GCTCCCTOGCTTGAACAGCAATT~CTCCTTTACA CCAGACCAMCTCAG +1850 +1900 GTCGCTCCACTGOOOACCATAOTCAGACCACCCCCCATCCTACCACCCCCCCCATAAAGTACCCA ,GAATGGAAACACATOAMkGCAAO +2000 FIG63 -lq -rn--mid 0 00 -1 t0L -100) *A AAkAAAA*A AkALAL AAAL LAALAA ~AAkAA*AAALA LLAAAALLAAAAAAAA A Aj*AAA, B -CClCGGGGACATG;AC CCCAG;AcAACG;AACGCA"ACAGGIV.A( IAGAGAGfCI'CAArI'CC'IAGCACAGGC-yCCACfG AlALAt *AAA k a ALA AALA ALAALALAALAAA *A A *ALALA 11 -GACaMK)A ACL(X'tI:--AGV'IG)GAG~rrA'rTCIACCLGC~CGG &ALALA A A A~h *LhAALA******LLALAAA kkLLALL LALL It -C~GAArA;ICCGC)-C(CCA.GA~XGCC~CLXACG~rA~'AICACGAAG-)7VGG k, A A A A k AAALAAALAAAAAA A*ALLALA tip!. -AAGGGAAcGAC AcAGCGGZGCKArICC--CAI~GAL-GGGAGGGCicrAMxA~rACACrGAAGCCCCI'CAG(GG *A LA*Ak*AAAALLLL A A AL*A*AAA A A*ALL*AL AAAAALLLLA AL *AAALAL fAIAAKITGATrAC.AGCACA-- CA(C.1GX(;L.AAGGGGAGAGAGAGAACCCAGVATAAAAAATGGCCCACAAGGACCAATIWCA AA LAAL~AAAAA A Alk A A, AALLAAA AA itLLL LAAALL*ALA AAAA *A A*A* LA A If CC--CC'1'GCKAAI~ACACAC;AA-A-- CAGGLXXXXGCAACAGIVCWAGAGA AGGGCCAGATAAAAAGGGCCCACAGAGACCGCI'CA *LA A AAA A AA A A*Ak A A At* &A LLLfiAAAAA*AAL *A*A AAL* *A A R -CitCAGCCATAATAAA'i~rATAGGA-AA(CAoGAG CCl GGaIcGAGGCAAACAGr-AGGTATAAAAAG GCGCr GGACCAGVCA ,A**AA LA*AALAL A AA lkA AAA*, A *A*A *A AL*A AALAAALAA ALAL A* A 31 P -,CANICCC-A(X>-ACCCA( ;&W:C.CAC;PcCA(:r('t'I;( CC1I'GACAkwhIACC- G&1G[IC RIC A *ALAALL *A*AAA *A &*AAA& A;AAAAAA LALALLLLAAAALAL ALA A B -G;GA~iCCCAGG-kCC-CA' -IriCACCA(;AC(;A('rCAA (.I'Cvflx(;ACAG;CVCACC-AGCIrATG ATGy **&AAAAAL AAA A A L*A AAAAA&AA*A*AAALA LAkL >~l 11 -(CIltCCAGG -CXCCAACICCCG(AACGACI'CPtXX.'j(:kikCAC-CCACCi'AGClCCA ATGFI 4c A A* L AAAL a A* AAA *&AAA LLAAAA*AL A AAk *AA R -GcA-cCCw'GAaCcCAt:'r-CCArClc(;-'iACa(-'I'ClAACAAI3CA&'GCIG ATrG A A ALA A *ALLA A tA ALL LAALAAA AAAAAk AL** ALLALL

11111. -CK)CAGWCA:'CCuCAfA;;,IC'I=A~4A~AIW NMG 0 RBS ,AGGAAA CAGACC A-T-G Gc i *GCA GCC AAGCTT.. PSI I Hindl1 NcoL, Psi 1, Hind III RBS .ACGAA CAGACO pre PGH met ala cla ATGIGCA GCT gly pro org GGC CCT CGG. Nco I Pst I Insert 800bp PstI fragment Eco RI Psi .M WO 8,8/08026 PIA8/00 PCT/AU&8/00109 6/11 RBS +24 pKTGH CACAGGAAACAGACC pGHXC.l CACAGGAAACAGACC ATO ATC 3 keG pGHX2.1 PGHiXF CACAGGAAACAGACC CACAGGAAACAGACC OCT ala TTrC phe GAG GAG ZT C phe ?he phe OCA 0CC CCT ala gly pro CCA GCC ATO pro ala me CAT CAT TAA GAT GAT AAC 7sp asp asa CCA GCC ATO pro ala met C CA GCC ATG pro ala met CCA GCC A TO0 pro ala me CCA 0CC 4GO pro ala me COO arg CCC pro ATG GOT pro CCO pro T C ser Tcc ser C CA pro CCA pro TCC ser T ser .CC ser GTG CTC CTG val leu leu AGC CTA TTT,.. ser leu phe 0CC 4TG CCC ala met pro 0CC ATO CCC ala me pro AOC CTA T TT. ser 1.eu phe AGC CTA T ZT... ser leu phe AOC CTr TC. ser leU phi pG~iXS .4 .CACAGGAOO-.TAATT AT G pOG{XS. 9 .~C4CAGGAG0T)AAAAT ATG pO C.CI CACAGGAAACAGACC A T met pGHXSC. 4 CACAGAGOT.AATAT AT G CCG CTG TCC ACC CTO pr'3 feu ser set leu TTC.I. phie FIG6 k EM WO 8/68026 PCr/AUSS/0O 109 7/11 200-.. 97.4- 68.0- 43.0- 25.7- 4. 18.4 14.3- FIG 7 r I SUBSTITUTE SHEET 0 0 CD hMT-IIA +1i +10j -120 +130 f-1-50 link er -130 +40 -150 1t60 MeL PGH cDNA FIG68 j VO P/08026 ~WO~8O8O26PCr/AU8/00109 9/11 1 2 3 4 5 6 7 8 9 10 11 B t* B 1 2 3 4 5 6 7 8 9 10 11 FIG 9 W1STITUTS SHEET m *WO 48/08026 PTA8/00 PCr/AU&8/00109 10/11 B C 64W m 4m FM~ SUBSTITUTE 3H-EET 0 Females FIG3 11 295 LLIIIOIlS Males I1a80 736 Iemacontrols malt) controls 375 739 6 0 12 14 16 6 8 10 12 14 Age (weeks) Age (eeks)Age (weeks) AM p F~* INTERNATIONAL SEARCH REPORT IenonaIlme Atiol-CalOn No PCT/AUS8/00109 1. CLASSIFICATION Of SUSJICT MArTW I 114-410 :tlaaC111o 1,M'OOs 1 3701" A.:A:t ,i ACCO~d1IO I* inernational Fslgfnt Clatsit'icatsart (IFIC) of 10 0011% National CIAllii~Cilon anid IPC int. C1. 4 C12N !5/00, C07G 17/00, C07H 21/04, C12'P 19/34 it. FIKLOS 119ANCM4D M,,..ium Cocumentlatio a rcnd Catia lsCo,0 SWutIM CIlsatialon Sno IPC WPI, WPIL, USPA (DERWENT DATABASES) :Keywords: "Transgenic" "Porcine Growth Hormone or PGH" Chemical Abstracts _Kevwdords "Tranqgeni" d "ICnW.__hon n OCU nlation Sa4vci..d alt,. than MIne.m en Oocurnintaton pig" and "Porcine to the Waent InaE SuCh OoCU nnt are Ife'i~dd r lots field& SOaICMed I Gr w h Ha x, AU :IPC as above PGH"I GenBank, NO.RF, EMBL, Kyoto Databases 11t. gCCUMINTS CONSIOILAIO TO 69 AILCVANTO Cata~o'~ CIsteen of Oacur,40nt, -11 indication, -nero sooeaoeialk, ol th. fulfacat 0668029.1 I~'eetto Cls.ee 4o, t X Hammer, R.E. et. al "Production of transgenic rabbits, 1,2,4,16, Y sheep and pigs by microinjpction", Nature', Volume 315, 17 issued 20 Jutie 1985, (Macmillan Journals Ltd, London, England) see pages 680 to 583. X Palmiter, R.D, et. al. 'ietallothionein-Human GN 1 Y Fusion Genes Stimulate G~rowth of Mice". Science, 16 Volume 222, issued 18 November 1983, (American Association for the Advancement of Science, Washtington USA, see pages 809 to 814, X Palmiter, R.D. et. al. "Dramatic qrowth of mice that 1 Y develop from eggs mWcroi-, Jacted with metallIothionei n- 26 growth hormone fusion genes". Nature, Volume 300, issued 16 December 1932, (Macm-illan Journals Ltd, London, England, see Paqes 61'. to 615, (continued.. Steial otoqitee.1 ocijr~nta* tiale 404- 3, 04%ot aftti t er e I,njromtoeie MOOe 441a ivew catgores a C144 dc~roritloi otiollit dat. and mot in COAnl.C% -4hi tfl e 'aO 4061 .t0 dciild iin trogn a' tt.o th e niefn i1t lIC to IAijdo"W"f the ofincioto of tnaory unclollf-Al A$ considered 10 Do 0 1 o,ciigo tiF1014in41141 sof saleo 40cuiin but 0 yalloned an Ofo t thi e r. nt.iltoil "go* dtUrnani 011 OoeMICU14i reva.nce nt tea r d invea~ iti" Fiin Gl CatJ~ 4 Ce cnaidored novel of cannot of constifar. 1o d*cufnflI .tt.CA n'Oy Ito-~a doiabit On gtiaeity cluffinl of no. nicnieam e~Iititga4 o talt In. oti a o a l ainother -Y 40cuerien of tlo'iiciur fiiavanca; the Claime~d in.o tieam ,Ito lio. or las 1 Of~ a 000 aV 14411 ae t 041 coannol to catseeea1 Voivlm0h or IMorsiv 004f $4 n doi. '0 dtitueniprlaig loo1 n 00al dobCIOsU'.o use, aetiti-0io er qurnefl C~ffloimied i~,o etc' mantintonl lUth tint M10A O.Ing avoiC o oulcon *40104 lit,4thn n oeshodl 110at hi tern atonl("n ot bu enato A Memer of %hie earpa talent hienily ly, ClIRIICATION i$o aln tIGIMe"toa otr lo oitto *1 lt ACIual COM04411ion t I ISO Intsa"notO S*S#c iA Oi.. alng0 i noraqntSrtl I. August 1988 (01.08.88) i" al Aulitoiila AUST1RALIAN PATENT OFFICE H.CA to,,i. .TlA~~leoi nolI...' Fish, y International Aopllcstion No. PCT/AU88/001C9 111, DOCUMENTS CONSIDERED TO ME RELEVANT (CONTINUED FROM THE SECOND SHEET) A aeoyCitation of Oocument, with irw-cation, wre appropriate, ofI th relevant passages Relevant to Claim No X EP,A, 177343(GENETECH, INC.) 9 April 1986 5106,15 (09.04.86) X 1van .der Putten, H. et. al ."Developmental fate of 1 a human insulin gene in a transgenic mouse". Molecular and General Genetics, Volume 198, issued 1984, (Springer-Verlag, Heidelberg, West Germany), I see pages 128 to 138. A Burki, K and A. Ullrich. "Transplantation of the human insulin gene 'into fertilized mouse eggs'. The 1 EMBO Journal, Volume 1, No.1 issuod 1982, (IRL Pres! Limited, Oxford, England), see pages 127 to 131. X WO,A, 82/04443 (OHIO UNIVEPSITY) 23 December 1982 1 (23.12.82) X Pursel, V.G. et. al. "Development of 1-cell 1,2,4 Y and pig ova after microinjection of genes", 16,17 Journal of Animal Science, Volume 65, Supplement 1, is-sued 1987, (American Society of Animal Science, Champaign, USA), see page 402 (Abstract). X AU,A, 19331/83 (BIOGEN 5 April 1984 5,6,15 Y (05.04.84) 16,17 X AU,A, 20929/83 (JOHN PETER ADELMAN nd 5,6,15 Y PETER HORST SEEBURG) 17 May 1984 '.05.84) 16,17 X Vize, P.D. and. J.R.E. Wells "Isolation and ,5,6,15 Y characterization of the oorcine growth hormone 16,17. ciene". Gene, Volume 55, issued 1987 'Elsevier Scientific Publishino Company, Amsterdam, NetherlemcQ see pages 339 to 344. Farm PCT'ISA'21IOW (eta hqeI) (JanuasY t285) I m U 7 U ANNEX TlO TBE 2=1ERTIONAL SEARCH REPRT ON UMENA-TIONATL APPLICATION NO. PCI'/AU 88/00109 This A~nnex lists the known "All publication level patent family Dnbers relating to the patent documents cited in the above-rrentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent Do~cument Cited in Search Patent Family Members Report AU 19331/83 DD 219505 DK 4400/83 EP 104920 ES 55948 ES 8504937 Th 69810 JP 59173083 NO 833464 NZ 205675 PT 77392 ZA 8307034 AU 20929/83 DK 5091/803 EP 111389 ES 527077 ES 8504255 JP 59144743 ZA 8308277 WO, 8204443 EP 81570 EP 177343 JP 61092575 US 4680262 END OF ANNEX 23@/208/1