NZ270923A - Increasing hair and wool production with a growth factor, dna therefor and transgenic animals - Google Patents

Description

2loci23 / .■ > i\ rev i ■: y V/ J'" 2 6 APR 1996 RECEIVED Patents Form No. 5 Our Ref: JP204459 NEW ZEALAND PATENTS ACT 1953 Complete After Provisional No. 270923 filed 11 April 1995 COMPLETE SPECIFICATION WOOL AND HAIR GROWTH AND CHARACTERISTICS We, LINCOLN UNIVERSITY, incorporated under the Lincoln College Act 1961 as amended by virtue of the Education Amendment Act 1989, of Ellesmere Junction Road, Lincoln, Canterbury, New Zealand hereby declare the invention, for which We pray that a patent may be granted to us and the method by which it is to be performed, to be particularly described in and by the following statement: PT0576770 (followed by page la) 27092J WOOL AND HAIR GROWTH AND CHARACTERISTICS The invention relates to wool and hair growth and the characteristics of such growth. More particularly the invention relates to stimulation of wool and hair growth and more particularly to increased wool growth in sheep. Improved wool production from transgenic sheep is shown.

The production of good quality wool is an important industry in many parts of the world. Hence any method by which wool production is increased would be a major benefit to the wool industry.

One way of achieving increased wool production is by breeding animals such as sheep and goats and selecting those species with wool production or characteristics. Unfortunately, this is a slow and unreliable process.

A method of increasing hair growth generally would also be of major benefit . In particular, there is a need in the cosmetics and/or pharmaceutical industries for ways of increasing human hair growth. Various creams, ointments, gels etc are available to stimulate and renew hair growth. Unfortunately they are often messy and/or unreliable.

It is therefore an object of this invention to go at least some way in overcoming at least some of the above mentioned problems by providing a method of stimulating hair and/or wool growth or to at least provide the public with a useful choice. (followed by page 2) The invention provides a transgene adapted for the production of a transgenic animal having increased hair or wool production compared with a non-transgenic animal of the same species and variety, the transgene including a growth factor gene under the control of a wool follicle-specific promoter.

The invention also provides a transgenic animal having increased hair or wool production compared with a non-transgenic animal of the same species and variety, the animal including a transgene according to the invention.

The invention also provides an embryo or zygote containing the transgene as above.

The invention also provides a method of producing transgenic animals with increased hair or wool production compared with non-transgenic animals of the same species and variety comprising inserting a transgene containing a growth factor under the control of a wool follicle-specific promoter into a suitable zygote or embryo.

The invention also provides a method of increasing hair growth or wool production comprising applying to the skin of a suitable animal an effective amount of a pharmaceutical^ acceptable composition comprising a growth hormone or growth factor produced from a transgene which increases or stimulates hair or wool growth.

In particular the invention provides a transgenic sheep having increased wool production compared with a non-transgenic sheep of the same species and variety.

The transgene may be a growth factor gene but is preferably an insulin-like growth factor (IGF) gene, such as IGF-1.

A wool follicle-specific promoter is the preferred promoter. The promoter KER is especially preferred.

The most preferred transgene is KER-IGF-1. Any suitable promoter (pm) may be used.

An embodiment of the invention will now be described, by way of example only and with reference to the accompanying drawings in which: Figure 1 shows the Xba I restriction sites in polGF-1; intellectual property office of n.z. 2 2 JUL 1998 RECEIVED 270923 IGF; Figure 2 shows the structure of the plasmid p2Fok; Figure 3 shows the plasmid pKER-IGF; Figure 4 shows the Xba I restriction sites in pKER- Figure 5 shows the IGF-1 fragment; Figure 6 shows pBluescript SK; Figure 7 shows the plasmid pIGF-AI; and Figure 8 shows wool production in first generation animals in the second year of life.

The aim of the study in the following example was to target expression of the sheep IGF-1 gene to the wool follicle of transgenic sheep with the aim of affecting wool production or properties.

The KER-CAT construct (Supplied by The Upjohn Company) was used to construct KER-IGF-1. A protocol was developed for year-round production of sheep pronuclear embryos and transgenic animals were produced by microinjection of DNA.

Construction of the KER-IGF transgene Recombinant DNA techniques used to construct the transgenes are generally known and are as described in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, (1989). Figures 1 to 3 show formulas of the plasmids and fragments used for the construction of the vector pKER-IGF. The structures represent both circular and linear double stranded DNA with transcription occurring from left to right (5' to 3'). Asterisk (*) represent the bridging of nucleotides to complete the circular form of the plasmids. 270923 Endonuclease restriction sites are indicated below the line by an arrow and the name of the specific restriction enzyme. Capital letters below the line are used to represent the fragment of interest for the various fragments of DNA depicted.

The recombinant promoter-growth factor gene construct, pKER-IGF is made by ligating a sheep IGF-1 cDNA behind the promoter region of the mouse ultra high sulfur keratin gene pUHSK-704Eco (McNab et al J. Invest.Dermatol. 92:263,1989). The plasmid poIGF-1 (Wong et al DNA 8:649, 1989), which contains the ovine IGF-1 cDNA (clone A6) cloned in pTZ18R-B (Pharmacia) is used. As shown in Figure 1, poIGF-1 is digested with restriction enzyme Xba I which has two recognition sites in poIGF-1, one upstream and one downstream from the IGF-1 cDNA. Thus, digestion with Xba I produces two DNA fragments, one of which contains the entire IFG-1 cDNA as well as 66 base pairs of vector sequence. The IGF-1 fragment is designated BBBBBBBB and the vector sequence is designated C in Figure 1.

The fragment generated is isolated from a 0.8% agarose gel in Tris-Borate-EDTA (TBE) buffer by running it into DEAE paper (Schleicher and Schuell). The DNA is then eluted from the paper in a buffer containing 50 mM Tris-Cl pH 8,1 M NaCl, and 10 mM EDTA pH 8, ethanol precipitated, washed with 70% ethanol and resuspended in TE buffer as described in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989). The purified fragment is then ligated into a plasmid named p2Fok (McNab et al Proc. Natl. Acad. Sci. USA 87:6848 1990) which consists of the promoter region of the PUHSK-704Eco gene (called KER and designated DDDDDDDDD in Figure 2) cloned into the Sma I site of plasmid pBluescript (Stratagene). 270923 p2Fok is digested with restriction endonuclease Xba I to open the plasmid at a site downstream from the KER promoter then treated with alkaline phosphatase to dephosphorylate the ends and prevent self ligation of the plasmid. The purified IGF-1 cDNA is ligated to the Xba I-digested p2Fok. The resulting plasmid is called pKER-IGF (Figure 3).

The ligation product is used to transform E. coli DH5<* according to the method described in Hanahan et al J. Mol. Biol. 166:557, 1983. The resulting bacterial colonies are grown individually in 10 ml Luria broth overnight and plasmid DNA is extracted from 1.5 ml of culture as described in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, (1989). The extracted plasmid is tested for the presence and orientation of the IGF-1 insert by digestion with restriction endonuclease Bam HI and ru;<ivlng the DNA in a 0.8% agarose gel.

Production of transgenic sheep Production of one-cell sheep zygotes The procedure described is performed all year-round. Donor animals are selected at random from a flock of 400 mixed-age Coopworth ewes maintained outdoors on rye grass/ white clover pasture. Six ewes undergo synchronisation of ovulation every week. Synchronisation is achieved by insertion of a progesterone-containing intravaginal device (CIDR: controlled internal drug release device, type-G, New Zealand Dairy Board) containing 300 mg progesterone, on Day-12, removing the CIDR and replacing it with another on 270923 Day-3, and removing the second CIDR on Day 0 (ie. 12 days of CIDR treatment). Superovulation involves twice-daily i.m. injection of follicle stimulating hormone (FSHp Schering Corporation), giving 5 mg and 4 mg on Day -2, 4 mg and 3 mg on Day -1, and 3 mg and 2 mg on Day 0, followed by a single i.m. injection of 80 g gonadotropin-releasing hormone (GnRH, Fertagyl, Intervet) 28h after removal of the second CIDR. Ewes remain on pasture during this period but are mustered into yards for each step in the procedure.

From Day 0, a vasectomised ram with a crayon harness is run with the ewes. Ewes which are marked by the vasectomised ram are inseminated 48h after CIDR removal with 250 1 of fresh semen placed in the body of the uterus by a laparoscopic technique.

Surgical recovery of the zygotes is performed 64 to 67h after CIDR removal. Ewes are anaesthetised with i.v. sodium pentobarbitone and the uterus horns and oviducts exteriorised via a mid-ventral laparotomy using sterile techniques. A polythene tube (5 cm x 1.8 mm o.d.) is inserted into the oviduct via the fimbria and the oviduct is flushed with 10 ml Dulbecco's phosphate buffered saline solution containing 5% BSA (Sigma) from a syringe with a blunt 20G needle inserted into the uterus near the uterotubal junction. Flushing is performed on both oviducts and the numbers of corpora lutea and eggs are recorded for each ewe.

Microinjection of DNA into one-cell sheep embryos Plasmid pKER-IGF is prepared essentially as described in Maniatis et al, Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, (1989), except that two rounds of cesium chloride purification are performed. The recombinant fragment containing the keratin promoter and 270923 the IGF-1 cDNA is removed from the plasmid pKER-IGF by restriction digestion with Kpn I and Sac I then purified from a 0.8% agarose gel by the glass milk binding method (described in Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1939) using a commercial kit (GeneClean, Bio 101), according to the manufacturer's instructions. The purified DNA is microinjected at a concentration of 4 ng/y1 into the pronucleus of one-cell sheep embryos according to the procedure described in Wagner et al, Proc. Natl. Acad. Sci. USA 78; 6376, 1981. The microinjected embryos are transferred into the oviduct of recipient ewes which are synchronised with the donors by inserting a CIDR and removing it on the same days as for the donors. At birth, every lamb undergoes a 0.5 cm2 ear biopsy from which DNA is extracted and used for Southern blotting using an IGF-1 cDNA radioactive probe, according to standard protocols (Maniatis et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989).

Analysis of IGF-1 expression in transgenic sheep skin by riboivuclease protection assay Construction of pIGF-A-1 pIGF-A-1 is contn ..ucted to serve as template for synthesis of a rib.^probe to be used for ribonuclease protection assay (RPA). It is made of 199 base pairs of sheep IGF-1 cDNA 5' sequence and 66 base pairs of pTZ18R-B vector sequence cloned in Bluescript.

As shown in Figure 4, pKER-IGF is digested with restriction enzymes Xba I which has two recognition sites in pKER-IGF, one upstream and one downstream from the IGF-1 cDNA. Thus, digestion with Xba I produces two DNA fragments, one of which contains the entire IGF-1 cDNA as well as 66 base pairs of vector sequence. 270923 The IGF-1 fragment is purified with DEAE paper as described above then digested with HphI (Figure 5).

The fragments generated by the Hph I digest are cloned into pBluescript SK (Stratagene). Plasmid pBluescript SK is a cloning vector which has a region that includes multiple cloning sites; (ie. a series of unique restriction enzyme cut sites), designated MCS in structure 6, flanked by binding sites for RNA polymerases T3 and T7 (designated T3 and T7, respectively, in Figure 6). DNA fragments to be subcloned are inserted in the vector at sites in the MCS and transcribed into RNA in vitro using T3 or T7 RNA polymerase. pBluescript is opened by restriction digest with Xba I. The linearised plasmid and the Hph I-digested IGF-1 cDNA are treated with T4 DNA polymerase and free nucleotides to form blunt ends then ligated. After transforming of E. coli DH5ot , and extraction of DNA from individual colonies, plasmids containing the 199 base pairs of 5' IGF sequence are identified by restriction digest with Sal I. Treatment with Sal I also indicates the orientation of the insert. This new plasmid is called pIGF-A-1 and is shown in Figure 7.

Analysis of IGF-1 expression in the skin Wool from an area of about 5 cm x 5 cm in the mid-side of transgenic sheep is clipped. After local anaesthesia with 2% Xylocaine, a 0.5 cm2 skin biopsy is taken using a trephine and kept in liquid nitrogen until processed. Total RNA is extracted from the skin biopsies by homogenising the samples with a Polytron tissue homogeniser (Brinkmann Instruments) in l ml of Trizol 270923 solution (Life Technolgies). After incubation at room temperature for 5 min, 0.2 ml of chloroform is added and the samples are centrifuged at 12,000 g for 15 min at 4°C to separate the RNA, which stays in the upper aqueous phase, from DNA and proteins. The upper phase is transferred to a new tube and the RNA is precipitated by mixing the aqueous phase with 0.5 ml isopropanol followed by centrifugation at 12,000 g for 10 min at 4°C. The RNA is then washed with 70% ethanol, dried, resuspended in 20pi H2O and analysed by ribonuclease protection assay (RPA).

Ribonucleaaa protection assay RPA consists of mixing the sample RNA with a radioactive probe complementary to the sequence to be detected followed by treatment with ribonuclease. If the target sequence is present, the probe will hybridise to it and form a double stranded molecule that is protected from digestion with ribonuclease, whereas the unhybridised probe is digested. The RNAse digested sample-probe mix is then run in a polyacrylamide gel and exposed to X-ray film where the protected fragment produces a band.

The probe is made by incubating pIGF-A-1 with T7 RNA polymerase, free nucleotides, and 32P UTP, using a Boehringer-Mannheim kit, according to the manufacturer's instructions. A radioactive antisense RNA fragment containing 199 base pairs of IGF-1 sequence and 66 base pairs of pTZ18R-B vector sequence is synthesised. This allows the resolution of the two RNA fragments in a polyacrylamide gel. 270923 RPA is performed using an Ambion kit, according to the manufacturer's instructions. Twenty yg sheep skin RNA and 105 cpm of IGF-1 antisense probe are mixed in hybridisation buffer (80% formamide, 100 mM sodium citrate pH 6.4, 300 mM sodium acetate pH 6.4, lmM EDTA) heated at 90°C to denature the RNA, incubated at 45°C overnight, and digested with 0.5 unit RNAse A and 20 units RNAse T1 in 220 1 at 37°C for 3 0 min. The RNAses are then inactivated and the protected RNA precipitated by adding 3 00 1 of the kit's inactivation/precipitation solution, freezing for 15 min at -20°C and centrifuging at 10,000 g for 15 min. The RNA pellet is dried and resuspended in gel loading buffer (80% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue, 2mM EDTA) and run in a 5% polyacrylamide gel in TBE containing 8 M urea at 200 V until the bromophenol blue reaches the bottom of the gel (approximately 90 min). The gel is then dried under vacuum and exposed to X-ray film overnight.

Wool analysis A mid-side patch is defined by clipping the wool over a 10 x 10 cm area in 3 month-old lambs. The animals are maintained outdoors on rye grass/white clover pasture with males and females in separate paddocks. Wool is clipped from the skin patches every three months, cleaned and weighed. When the sheep reach the age of 15 months, fleece weight, fibre diameter, staple strength, bulk, and fibre medullation are recorded by techniques well known, to those skilled in the art. 270923 Transgenic Integration, Inheritance, and Expression The performance data are reported in Table 1. Phase I refers to experiments with KER-CAT, Phase II with KER-IGF-1. With KER-CAT, 4/31 (12.9%) lambs born were transgenic, compared to 5/103 (4.9%) for KER-IGF-1. This difference is probably within the variation to be expected from the method, but could be associated in some way with the difference in the constructs. There was a substantial increase in the pregnancy rate in Phase II. The overall performance is comparable to international standards in producing transgenic sheep.

Skin expression in Phase I was examined by CAT assay. Of the founders (Gq), 1/4 showed expression in skin. Preliminary data from in situ hybridisation indicate that the expression in this ram is confined to the keratogenous zone of the follicle, consistent with findings in the mouse (McNab et al, above). This and another Gq ram were mated to non-transgenic ewes and 71 Gi progeny were born. In one case 45% of the offspring were transgenic and in the other 40% were transgenic, approximating expected Mendelian inheritance. Line 1, from the ram expressing the transgene, had 35% penetrance of expression, whereas in Line 3, from the non-expressing ram, none of 17 lambs tested showed expression (Table 2). An "organ recital" of CAT activity in different tissues from 2 G]_, Line 1 progeny showed that expression was substantially confined to the skin. The level of CAT activity in the skin followed the seasonal pattern of wool growth.

Similar studies were carried out with the Phase II transgenics. Of the 5 founder animals, 2 showed expression of IGF-1 in skin biopsies. Cellular localisation and tissue specificity studies are in progress. A progeny test was done with the sole transgenic male, creating Line S in which 51% of lambs inherited the gene and 80% showed expression (Table 3). 270923 Wool Production and Properties A progeny test was carried out on animals of Line 9, in which wool growth, production, and fibre characteristics were assessed in 19 transgenic lambs and compared with 22 non-transgenic or non-expressing transgenic half-sib controls. Pooling of data from non-transgenic controls with non-expressing transgenics was done after analysis of wool growth revealed that there was no significant difference between them and that both were significantly different from transgenic expressors. The animals were kept under conventional New Zealand husbandry practices on a rye grass / white clover paddock, with no supplemental feeding or special precautions (apart from approved containment for transgenic sheep). The rams were intact and the ewes were run dry.

Data on clean fleece weight (g/kg body weight) at hogget shearing are presented in Table 4. Overall, there was a 10% increase in transgenic compared with control animals (p=0.028) and the effect was more pronounced in rams (12.5%, p = 0.004) than in ewes (4.6% p > 0.05). We have no explanation at present for this sex difference. To allow for differences in birth date, the data were also expressed as rate of wool growth (clean fleece weight /days of age). As shown in Table 5, transgenic animals had a significantly faster rate of wool growth (p = 0.021), with the effect being more pronounced in males.

Expressing the wool growth in g/day is justified by the absence of a significant difference in hogget body weight and rate of weight gain between transgenic and control animals (Tables 6 and 7). In fact, the means for transgenics were slightly lower than for controls. As would be expected, ram hoggets were significantly heavier than ewe hoggets (p = 0.051, Table 6). 270923 Fibre diameter at hogget shearing in these coarse-wool (Coopworth) sheep showed no significant difference between transgenic status or between the sexes (Table 8).

Wool Production in First Generation Animals in the Second Year of Life (1995) Figure 8 shows wool production in first-generation animals in the second year of life (1995). Transgenics maintain an advantage over non-transgenics. For the second generation, the transgene was inherited by 28 out of 63 progeny (44%) .

It is to be understood that the invention is not restricted to the above embodiment but that numerous variations and modifications may be made without departing from the scope of the invention as set out in this specification.

For example, the invention is not limited to sheep but may include, for example wool from goats. Also, there is no restriction to the use of IGF-I as the gene promoting hair/wool stimulation; any gene which promotes stimulation of hair/wool is envisaged. 270923 Table 1: TRANSGENIC SHEEP PRODUCTION ' Phase I Phase II KER-CAT KER-IGF1 Embryos collected 796 1225 Embryos/donor 3.9 6.6 Embryos injected 484 (61%) 716 (58%) Embryos transferred 371 (77%) 591 (83%) No. Recipients 76 116 Embryos/recipient 4.9 .1 No. pregnancies 23 (30%) 72 (62%) No. Lambs born 31 103 No. Weaned 22 (71%) 87 (85%) No. Transgenic 4 Transgenic % of births 12.9 4.9 -_Transgenic % transfers 1.1 0,8 Transgenic % of injected 0.8 0.7 Table 2: EXPRESSION AND INHERITANCE OF KER-CAT 1. Transgenic Founders No.

Sex Born Expression 1 M 3/4/91 Y 2 F /4/91 N 3 M 26/6/91 N 4 F 26/7/91 N 2. Progeny Test Ram No. Ewes No. Lambs No. Transgenic No. Expressing 1 42 80 . 36 9/26 3 29 57 23 0/17 Table 3: 270923 EXPRESSION AND INHERITANCE OF KER-IGF1 1. Transgenic Founders « No.

Sex Born Expression F 28/10/91 N 7 F /11/91 Y 318 F 7/12/91 N 9 M 16/3/91 Y F 21/4/92 N 2. Progeny Test - Ram No. Ewes No. Lambs No. Transgenic No. Expressing 9 43 85 43 /25 Table 4 CLEAN FLEECE WEIGHT AT HOGGET SHEARING (g/kg body weight) Control Transgenic Mean %5 Male 55.0 63.0 59.5* 12.50 ± 1.7 ± 1.9 ±1.3 Female 61.6 66.2 63.9* ' 4. SO ± 1.8 ± 2.3 ±1.4 Mean 58.8b 64.6" 9.86 ±1.2 ±1.5 a p = 0.028 b p = 0.004 Table 5 RATE OF WOOL GROWTH (g/day) Control Transgenic Mean %5 Male 9.4 .5 9.9 11.7 ±0.3 ±0.3 ±0.2 Female .0 .4 .2 4.0 ±0.3 ±0.4 ±0.2 Mean 9.7" .5* 8.2 ±0.2 ±0.2 a p = 0.021 27°923 Table 6 HOGGET BODY WEIGHT (kg) Control Transgenic Mean %5 Male 75.3 74.0 74.7* -1.7 ±1.3 ±1.5 ±1.0 Female 73.1 70.4 71.7" -3.7 ±1.4 ±1.7 ±1.1 Mean 74.2 72.2 -2.7 ±0.9 ±1.1 a p = 0.051 Table 7 RATE OF BODY WEIGHT GAIN (g/day) Control Transgenic Mean' %S Male 168.6 ±2.9 165.8 ±3.4 167.2 ±2.2 -1.7 Female 163.9 ±3.1 153.0 ±3.9 161.0 ±2.5 -3.6 Mean 166.3 ±2.2 161.9 ±2.6 -2.1 Table 8 HOGGET FIBRE DIAMETER (microns) Control Transgenic Mean %5 Male 42.49 ±0.69 43.46 ±0.78 42.98 ' ±0.52 2.3 Female 41.42 ±0.72 43.26 +0.93 42.34 ±0.59 4.4 Mean 41.96 ±0.50 43.36 ±0.61 3.3 All tables give means ± s.d. 27 17

Claims (21)

WHAT WE CLAIM IS:

1. A transgene adapted for the production of a transgenic animal, the transgenic animal having increased hair or wool production compared with a non-transgenic animal of the same species and variety, the transgene including a growth factor gene under the control of a wool follicle-specific promoter.

2. A transgene as claimed in claim 1 in which the gene is an insulin-like growth factor gene.

3. A transgene as claimed in claim 2 in which the gene is IGF-1.

4. A transgene as claimed in claim 1 in which the wool follicle-specific promoter is KER.

5. A transgene which is KER-IGF-1.

6. A transgenic animal having increased hair or wool production compared with a non-transgenic animal of the same species and variety, the transgenic animal including a transgene as claimed in any one of claims 1-5.

7. An embryo containing a transgene as claimed in any one of claims 1-5.

8. A zygote containing a transgene as claimed in any one of claims 1-5.

9. A vector containing the transgene of any one of claims 1-5.

10. A method of producing a transgenic animal, the transgenic animal having increased hair or wool production compared with a non-transgenic animal of the same species and variety comprising inserting a suitable transgene into a suitable embryo or zygote, wherein the transgene is as defined in any one of claims 1-5.

11. A method according to claim 10 in which the transgenic animal is a sheep.

1 2. A method according to claim 10 in which the transgenic animal is a goat. 270 18

13. A transgene adapted for the production of a transgenic sheep, the transgenic sheep having increased wool production compared with a non-transgenic sheep of the same species and variety, the transgene including a growth factor gene under the control of a wool follicle-specific promoter.

14. A transgene adapted for the production of a transgenic goat, the transgenic goat having increased wool production compared with a non-transgenic goat of the same species and variety, the transgene being as defined in any one of claims 1-5.

15. A transgenic sheep having increased wool production compared with a non-transgenic sheep of the same variety and species, the sheep including a transgene as claimed in any one of claims 1-5.

16. A transgenic goat having increased wool production compared with a non-transgenic goat of the same variety and species, the goat including a transgene as claimed in any one of claims 1-5.

17. An embryo as claimed in claim 7 which is a sheep embryo.

18. A zygote as claimed in claim 8 which is a sheep zygote.

19. A transgene substantially as herein described with reference to Figures 1-4.

20. A transgenic sheep substantially as herein described with reference to Figures 1-8 or Tables 1-8.

21. A method of producing a transgenic sheep substantially as herein described with reference to Figures 1-8 or Tables 1-8. LINCOLN UNIVERSITY By Its Attorneys BALDWIN SHELSTON WATERS ASPEC13006 intellectual property office of n.z. I 2 JUL 1598 END OF CLAIMS