WO1999009817A1

WO1999009817A1 - Use of mariner transposan in the production of transgenic animals

Info

Publication number: WO1999009817A1
Application number: PCT/GB1998/002517
Authority: WO
Inventors: Helen Sang; David John Finnegan
Original assignee: Biotechnology And Biological Sciences Research Council
Priority date: 1997-08-22
Filing date: 1998-08-21
Publication date: 1999-03-04
Also published as: CA2300972A1; AU8817798A; JP2001513336A; EP1006790A1

Abstract

A method for the preparation of a transgenic animal embryo comprising the step of introducing a mariner-like element (MLE) containing a transgene into an animal embryo cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encoding a transposase. The resulting embryo may be used to generate further embryos or be allowed to develop into an animal. The invention is useful in introducing foreign DNA into selected animals.

Description

USE OF MARINER TRANSPOSAN IN THE PRODUCTION OF TRANSGENIC ANIMALS

The present invention relates to a method for introducing a transgene into an animal embryo and to the preparation of a transgenic animal therefrom.

Introduction of foreign DNA by microinjection into the newly fertilised egg, before the first embryonic cell cleavage, has become an established method for the production of transgenic animals. The foreign DNA is incorporated into the chromosomes of the animal and inherited as a stable additional sequence by the offspring of the founder transgenic animal.

The frequency at which this chromosomal integration occurs varies between species. The frequency is also influenced by the site of injection. If the DNA is introduced into one of the pronuclei of the fertilised egg, the frequency of production of transgenic animals is generally higher.

Transposable elements are defined sequences of DNA that can transpose to different sites in the genome of an organism. Transposable elements can be divided into several different classes, defined by the mechanism which they use to move from one genomic site to another. The ability of transposable elements has been modified to enable use of a transposable element from a particular species to be used as a vector to introduce foreign DNA into the genome of that species, e.g. the P element from Drosophila melanogaster is widely used to transform D. melanogaster (Rubin, G. M., & Spradling, A. C, Science 218 348-353 (1982) and US-A-4670388).

A method for culturing the chick embryo from the new ly fertilised egg to give hatched chicks has been developed and is described in EP-A-0295964 and in Perry. M. M. Nature 331 70-72 (1988). Subsequently, a method for injecting DNA into the cytoplasm of the chick zygote, i.e. the germinal disc, was described in Sang, H. M. and Perry, M. M., Mol. Reprod. Development 1 98-106 (1989) and in Perry et al ROILX'S Archive of Developmental Biology 200 312-319 (1991). The use of these techniques in the production of a transgenic bird was reported in Love et al Bio/Technology 12 60-63 (1994). However, continued use of this procedure for the production of transgenic birds has shown that the frequency at which transgenic birds can be obtained, defined by the incorporation of foreign DNA which is transmitted to their offspring, is low: 3 germline transgenic birds from a total of 254 live chicks were transgenic.

The efficiency of this method is therefore relatively poor with only 1% or less of chicks hatched after DNA injection have incorporated the injected DNA into their genome. Additionally, from each of these transgenic birds only a single transgenic line has been obtained, i.e. the transgenic offspring contain a few copies of the foreign DNA at a single chromosome site.

Injection of DNA constructs into the cytoplasm is also very inefficient in mammals when compared to pronuclear injection (Brinster et al Proc. Nat'l. Acad. Sci. USA 82 4438-4442 (1985)). Also most transgenes produced by pronuclear injection consist of an array of multiple copies of the injected DNA construct. The organisation of these arrays can have negative effects on expression of the transgene, e.g. reduce the level of expression or affect the tissue-specificity of expression.

Transgenic birds have also been produced using infection of retroviral vectors (Bosselman et al Science 243 533-535 (1989)). However, the use of retroviral vectors has several disadvantages. The risk of recombination of viral vectors with wild type retroviruses which are widespread in poultry populations is perceived as the most serious problem. Retroviral vectors are also very complicated to work with and are restricted in their capacity to incorporate constructs greater than approximately 8 kilobases of DNA.

The transposable element mariner was originally discovered in the genome of Drosophila mauritiana but closely related elements have been discovered in a wide variety of species both vertebrate and invertebrate (Robertson, H. M., Nature 362 241 (1993)) It has also been used to investigate pathogenic orgamsms such as Leishmama (Gueiros-Filho, F J., & Beverley, S. M , Science 276 1716-1719 (1997) and Haiti, D L., Science 276 1659-1660 (1997) However, the use of mariner descπbed by Gueiros-Filho et al and Haiti only relate to its use as a genetic tool, i.e for msertional mutagenesis and not as a means for the preparation of transgenic animals by transgenesis or mutagenesis. In summary, mariner has not been descπbed as being suitable for a role m the production of transgenic animals and nor is such a use contemplated in the pπor art

It has now been found surprisingly that the mariner element can be used as a vector in the preparation of transgenic animals

According to a first aspect of the present invention there is provided a method for the preparation of a transgenic animal embryo composing the step of introducing a mariner-like element (MLE) containing a transgene into an animal embryo cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encodmg a transposase

The term transgenic is used m the context of the present invention to descπbe animals which have stably incorporated a sequence of foreign DNA introduced by the mariner-like element (MLE) into their chromosomes such it that may passed on to successive generations of transgenic descendant animals In such circumstances, the initial transgenic animal is known as a "founder" animal The founder animal may have the foreign DNA or transgene incorporated in all of its cells or a sufficient proportion such that its progeny stably inheπt the transgene Where the transgene is only present in a proportion of cells of the animal, the animal is referred to as a chimera The present invention also extends to animals which incorporate the transgene stably or directly into their chromosomes and which express the transgene m their somatic cells without passing the gene onto their offspπng It should be noted that the term "transgenic", in relation to animals, should not be taken to be limited to referring to animals containing in their germ line one or more genes from another species, although many transgenic animals will contain such a gene or genes. Rather, the term refers more broadly to any animal whose germ line has been the subject of the introduction of a warmer-like element (MLE). So, for example, an animal in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a transgenic animal for the purposes of this invention as much as an animal to whose germ line an exogenous DNA sequence has been added.

In principle, the invention is applicable to all animals, including birds, such as domestic fowl, amphibian species and fish species. In practice, however, it will be to non-human animals (warm-blooded vertebrates), especially (non-human) mammals, particularly placental mammals, and birds, particularly poultry, that the greatest commercial useful applicability is presently envisaged. It is with ungulates, particularly economically important ungulates such as cattle, sheep, goats, water buffalo, camels and pigs that the invention is likely to be most useful. Of the avian species, the invention has particular application to poultry, including domestic fowl Gallus domesticus, turkeys and guinea fowl. It should also be noted that the invention is also likely to be applicable to other economically important animal species such as, for example, horses, llamas or rodents, e.g. rats, mice or rabbits.

The method of the present invention is directed towards the introduction of foreign DNA or a transgene into an animal embryo cell. The embryo cell may be at the single cell stage immediately following fertilisation which is the zygote stage. However, the introduction may be into an embryo cell from a later stage of embryonic development, e.g. from a 2-cell, 4-cell, 8-cell, 16-cell, 32-cell, or 64-cell stage embryo, or from an even later stage. A founder transgenic animal produced from such a later stage embryo may therefore be a chimera but its offspring can be selected for the presence of the transgene in all cells. The manner-hke element may be the transposable element mariner from Drosophila mauritiana or a closely related element from another vertebrate or invertebrate species (Robertson, H M , Nature 362 241 (1993)) The manner-hke element may conveniently be deπved from the cells of the animal whose chromosomes are to altered A nucleotide sequence which is a mariner-like element can be defined by its ability to act as a transposable element when introduced into a cell

Manner-hke transposable elements are about l,300bp long with terminal inverted repeats of about 30bp Each manner-hke element encodes a polypeptide that is a putative transposase and that has, on average, 34% ammo acid sequence identity with the polypeptides encoded by other manner-hke elements The am o acid sequences of the putative transposases of all manner-like elements include a characteπstic motif known as D, D34D, where "D' represents an aspartate residue The third aspartate of this motif is followed immediately by a tyrosme residue (Robertson, H M , J Insect Physiol 41 99-105 (1995))

The transgene can be contained in the manner-hke element at any point withm the mariner sequence Without being bound by theory, it is believed that approximately the final 100 bases of each end of the marine/ -like element may be important for function of the MLE and its incorporation into the chromosomes of a cell Thus the transgene may be positioned anywhere withm in the MLE except less than approximately 100 bases from each end The transgene may also replace the central sequences of the MLE, with only the ends of the element being retained

The transgene sequence contained m the manner -like element may be any desired foreign gene sequence Particularly preferred gene sequences include, but are not limited to. those gene sequences coding for proteins which are therapeutically useful, such as enzymes, hormones or other functionally active proteins, e g immunoglobulins, haemoglobin, myoglobm, cytochromes, etc Other gene sequences may encode proteins whose genes are absent or mutated such that the corresponding protein is not produced or is not produced in active form, 1 e. genes responsible for disease conditions such as cystic fibrosis or muscular dystrophy

Typically, the transgene sequences will also contain promoter sequences to direct expression of the transgene in a selected tissue, e g the mammary gland for secretion in the animals milk, in the yolk or albumen of an egg, or in the blood Further applications, include the expression of regulatory proteins that control immune rejection such that the organs of the transgenic host ammal may be used in Xenotransplantion into a recipient which is allogemc for the immune proteins being expressed m the cells Similarly, allotransplantation is also included

In agπcultural applications of the method of the present invention may be used to produce improved farm animals The transgenes introduced into the animals may include, but are not limited to, disease resistance genes, growth enhancing genes or genes which provide for improved charac ten sties in a particular trait or introduction of a novel trait

The introduction of the manner-hke element (MLE) may convemently be achieved by injection of the MLE into the cytoplasm or into the pronucleus of a zygote or the nucleus of an ammal embryo cell Other routes of introduction, such as electroporation or hposomes may be equally effective and used m the method according to the present invention

The MLE may be introduced m the form of a construct compπsing the DNA sequence oϊ & manner-hke element and the desired transgene or simply the nucleotide sequence of a manner-hke element and the desired transgene itself may be introduced Where a vector based method of introduction is used, the construct may be a plasmid, a cosmid or an artificial chromosome such as a Yeast Artificial Chromosome (YAC) or a Bacteπal Artificial Chromosome (BAC) The constructs may also contain additional regulatory sequences, if required, such as promoters or enhancers, depending of the foreign DNA being mtroduced A further aspect of the present invention is therefore a construct or a vector compπsing a manner-hke element containing a transgene as descπbed above In general, the MLE will be cloned in a plasmid vector for ease of manipulation and clomng-m of transgenes It may also be preferable to have the MLE vector in a circular molecule so that the DNA will be supercoiled to facilitate transposition

At the time of introduction of the manner-hke element, or shortly before or shortly thereafter, the method of the present mvention may contain an additional optional step of introducing exogenous transposase protein, or a DNA or RNA sequence encoding a transposase

The manner-hke element and the transposase introduced into the cell may be deπved from the same ammal species or different species

According to a second aspect of the present invention there is provided a method for the preparation of a transgenic animal embryo compπsing the step of introducing a mannei -like element (MLE) contaimng a transgene into an animal adult cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encoding a transposase

According to a third aspect of the present invention there is provided a method for the preparation of a transgenic animal embryo compπsing the step of introducing a manner-hke element (MLE) contaimng a transgene into an animal foetal cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encodmg a transposase

In any one of the methods accordmg to the first, second and third aspects of the present invention, the resulting animal embryo may be prepared by (l) removing the nucleus of the cell following introduction of the manner- ke element containing the transgene and its insertion mto the chromosomes, and (n) subsequently introducing the nucleus mto an enucleated oocyte which is allowed to develop mto an animal embryo. There are several methods described for the preparation of an animal embryo using nuclear transfer techniques and preferred techniques include, but are not limited to those described in WO-A-9607732, WO-A-9707669 and WO-A- 9707668.

In the method of the invention described above, a nucleus is transferred from a donor cell to a recipient cell. The use of this method is not restricted to a particular donor cell type. The donor cell may be as described in Wilmut et al Nature 385 810 (1997); Campbell et al Nature 380 64-66 (1996); or Cibelli et al Science 280 1256-1258 (1998). All cells of normal karyotype, including embryonic, foetal and adult somatic cells which can be used successfully in nuclear transfer may in principle be employed in a method according to the present invention. Foetal fibroblasts are a particularly useful class of donor cells. Generally suitable methods of nuclear transfer are described in Campbell et al Theriogenology 43 181 (1995), Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et al Biol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat'l. Acad. Sci. USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274. WO-A-9807841, WO-A-9827214, WO-A-9003432, US-A-4994384 and US-A- 5057420. The invention therefore contemplates the use of an at least partially differentiated cell, including a fully differentiated cell. Donor cells may be, but do not have to be, in culture and may be quiescent. Nuclear donor cells which are quiescent are cells which can be induced to enter quiescence or exist in a quiescent state in vivo. Cultured bovine primary fibroblasts, an embryo-derived ovine cell line (TNT4), an ovine mammary epithelial cell derived cell line (OME) from a 6 year old adult sheep, a fibroblast cell line derived from foetal ovine tissue (BLWFl) and an epithelial-like cell line derived from a 9-day old sheep embryo (SECl) are described in WO-A-9707669 and WO-A-9707668. A class of embryo-derived cell lines useful in the invention which includes the TNT4 cell line described in WO 96/07732. Cultured inner cell mass (CICM) cells are described in WO-A9737009 and WO-A- 9827214 and embryonic or stem-like cell lines are described in WO-A-9807841. Transgenic bovine fibroblasts for use as nuclear donors are described in Zawada et al (Nature Medicine 4 (5) 569-574 (1998) and in Cibelli et al (Science 280 1256-1258 (1998))

Where the donor cells are descπbed as being quiescent, such cells may not be actively proliferating by means of the mitotic cell cycle The use of a quiescent donor cell is descπbed m WO-A-9707669 The mitotic cell cycle has four distinct phases, Gl, S. G2 and M The beginning event in the cell cycle, called start, takes place m the Gl phase and has a unique function The decision or commitment to undergo another cell cycle is made at start Once a cell has passed through start, it passes through the remainder of the Gl phase, which is the pre-DNA synthesis phase The second stage, the S phase, is when DNA synthesis takes place This is followed by the G2 phase, which is the peπod between DNA synthesis and mitosis Mitosis itself occurs at the M phase Quiescent cells (which include cells in which quiescence has been induced as well as those cells which are naturally quiescent, such as certain fully differentiated cells) are generally regarded as not being m any of these four phases of the cycle, they are usually descπbed as bemg in a GO state, so as to indicate that they would not normally progress through the cycle The nuclei of quiescent GO cells have a diploid DNA content

Cultured cells can be induced to enter the quiescent state by various methods including chemical treatments, nutπent depnvation, growth inhibition or manipulation of gene expression Presently the reduction of serum levels m the culture medium has been used successfully to induce quiescence m both ovme and bovine cell lmes In this situation, the cells exit the growth cycle dunng the Gl phase and arrest, as explained above, m the so-called GO stage Such cells can remain m this state for several days (possibly longer depending upon the cell) until re- stimulated when they re-enter the growth cycle Quiescent cells arrested in the GO state are diploid The GO state is the pomt m the cell cycle from which cells are able to differentiate On quiescence a number of metabolic changes have been reported and these include monophosphorylated histones, ciliated centπoles, reduction or complete cessation m all protein synthesis, increased proteolysis, decrease m transcπption and increased turnover of RNA resulting m a reduction m total cell RNA, disaggregation of polyribosomes, accumulation of inactive 80S ribosomes and chromatin condensation (reviewed Whitfield et al, Control of Animal Cell Proliferation, 1 331-365 (1985)).

Many of these features are those which are required to occur following transfer of a nucleus to an enucleated oocyte. The fact that the GO state is associated with cell differentiation suggests that this may provide a nuclear/chromatin structure which is more amenable to either remodelling and/or reprogramming by the recipient cell cytoplasm. In this way, by virtue of the nuclear donor cells being in the quiescent state, the chromatin of the nuclei of the donors may be modified before embryo reconstitution or reconstruction such that the nuclei are able to direct development. This differs from all previously reported methods of nuclear transfer in that the chromatin of donor cells is modified prior to the use of the cells as nuclear donors.

The recipient cell to which the nucleus from the donor cell is transferred may be an oocyte or another suitable cell. A preferred class of recipient oocyte is described in WO-A-9707668.

Recipient cells at a variety of different stages of development may be used, from oocytes at metaphase I through metaphase II, to zygotes and two-cell embryos. Each has its advantages and disadvantages. The use of fertilized eggs ensures efficient activation whereas parthenogenetic activation is required with oocytes (see below). Another mechanism that may favour the use of cleavage-stage embryos in some species is the extent to which reprogramming of gene expression is required. Transcription is initiated during the second cell cycle in the mouse and no major changes in the nature of the proteins being synthesised are revealed by two- dimensional electrophoresis until the blastocyst stage (Howlett & Bolton J Embryol. Exp. Morphol. 87 175-206 (1985)). In most cases, though, the recipient cells will be oocytes. It is preferred that the recipient be enucleate While it has been generally assumed that enucleation of recipient oocytes in nuclear transfer procedures is essential, there is no published expeπmental confirmation of this judgement The oπgmal procedure descπbed for ungulates involved splitting the cell mto two halves, one of which was likely to be enucleated (Willadsen Nature 320 (6) 63-65 (1986)) This procedure has the disadvantage that the other unknown half will still have the metaphase apparatus and that the reduction in volume of the cytoplasm is behe\ed to accelerate the pattern of differentiation of the new embryo (Eviskov et al Development 109 322-328 (1990))

More recently, different procedures have been used in attempts to remove the chromosomes with a minimum of cytoplasm Aspiration of the first polar body and neighbouring cytoplasm was found to remove the metaphase II apparatus m 67% of sheep oocytes (Smith & Wilmut Biol Reprod 40 1027-1035 (1989)) Only with the use of DNA-specific fluorochrome (Hoechst 33342) was a method provided by which enucleation would be guaranteed with the minimum reduction in cytoplasmic volume (Tsunoda et al , J Reprod Fertil 82 173 (1988)) In livestock species, this is probably the method of routine use at present (Prather & First J Reprod Fertil Suppl 41 125 (1990), Westhusin et al , Biol Repiod (Suppl ) 42 176 (1990))

There ha\ e been very few reports of non-mvasive appioaches to enucleation m mammals, whereas in amphibians, irradiation with ultraviolet light is used as a routme procedure (Gurdon 0 J Microsc Soc 101 299-311 (I960)) There are no detailed reports of the use of this approach in mammals, although dunng the use of DNA-specific fluorochrome it was noted that exposure of mouse oocytes to ultraviolet light for more than 30 seconds reduced the developmental potential of the cell (Tsunoda et al , J Reprod Fertil 82 173 (1988))

According to a fourth aspect of the present invention there is provided a method for the preparation of an ammal, the method compπsing the steps of (a) prepanng an embryo according to any of the preceding aspects of the present invention,

(b) causing an ammal to develop to term from the embryo, and

(c) optionally breeding from the animal so formed

The ammal embryo prepared m accordance with this aspect of the present invention may be further manipulated pπor to full development of the embryo This may include the introduction of additional genetic matenal or to assay the embryo for particular genetic characteπstics or the presence or absence of a gene It is also possible that more than one ammal can be denved from the embryo where the cells of the embryo are used to prepare more than one embryo allowed to develop to term

The present invention therefore also extends to an ammal prepared by a method according to the fourth aspect of the invention

According to a fifth aspect of the present invention there is provided the use of a manner-like element m the therapy of a disease condition caused by the absence of a gene or the mutation of a gene This aspect of the invention also extends to the use of a manner-like element m the preparation of an agent for the prophylaxis or treatment of a disease caused by the absence of a gene or the mutation of a gene Such methods of treatment may compnse the introduction of a manner-hke element contaimng a transgene mto an ammal cell Where transposase protein, or a DNA or RNA sequence encoding transposase is also to be introduced, this step may be simultaneous, sequential or separate to the introduction of the MLE

The present invention therefore also extends to the preparation of an embryo according to any one of the preceding aspects of the invention m which the cells of the embryo are used in the treatment of a disease condition associated with the absence of a gene or the mutation of a gene Such cells may also be used to treat disease conditions in which the patient's cells are no longer active or effective especially neurological or hormonal disorders Preferred features for the second and subsequent features are as for the first aspect mutatis mutandis

The present invention will now be descπbed with reference to the accompanying Examples and drawings which are included for the purposes of example only and are not to be construed as being limiting on the present invention In the following Examples, reference is made to a number of drawings m which

FIGURE 1 shows PCR analysis of DNA extracted from embryos and chicks that survived for at least 12 days of incubation after injection of the røαrzMer-containing plasmid pMosl FIGURE 1(a) shows a diagram of pMosl, indicating sequences identified by PCR and unique restπction sites FIGURE 1(b) shows a graphical presentation of the results given m Table 1 The estimated copy number per genome equivalent of a lysozyme gene construct, injected m a previous seπes of expeπments (n=186), is compared to the estimated copy number of mat inei sequence The results of injecting pMosl with and without the addition of recombinant-deπved transposase protein are compared

FIGURE 2 shows a Southern blot analysis of genomic DNA isolated from individual Gi transgenic chicks, hybridised with a manner probe FIGURE 2(a) shows BamHI/Hindlll digests of individual chicks which each have a novel pattern of mαπ«er-hybπdιsιng fragments (lanes 1 to 7), and of the parent Go cockerel (lane 8) A control digest of non-transgemc chick DNA was run in lane 9 FIGURE 2(b) shows EcoRI digests of samples from the same birds as in FIGURE 2(a) The Band arrowed in lane 2 is the EcoRI fragment cloned in pZAP13 (see FIGURE 3)

FIGURE 3 shows the characteπsation of a single integrated marinei element FIGURE 3(a) shows the Southern blot of genomic DNA from individual Gi chicks digested with BamHI and Hindlll (from FIGURE 2(a)) was stripped and reprobed with the EcoRI insert from pZAP13. Hybridisation to a range of restriction fragments can be seen in all the samples, including the negative control (lane 9). FIGURE 3(b) shows a comparison of the sequence across the left and right ends of the mariner element in pMosl and pZAP13.

FIGURE 4 shows PCR analysis for present of the Tet^R gene in DNA from embryos and chicks that survived for at least 12 days of incubation after introduction of pMoslTet. The copy number of the Tet^R gene was estimated as described in the "Materials and Methods" and the results of co- injection of transposase protein compared with injection of plasmid alone.

Example 1 : Preparation of Transposase The mariner transposase used in the following experiments was purified from E. coli strain BL21 DE3 (Studier et al Methods in Enzymology 185 60-89 (1991)) carrying the plasmid pBCPMosl. This was derived from the expression vector pBCP368 (Velterop et al Gene 153 63-65 (1995)). The complete coding sequence of mariner transposase from the element Mosl was inserted at the Ndel site of pBCPMosl. These cells were grown in Luria broth (LB) in an orbital shaker (200rpm, 37°C) to an OD₅₅₀ of 0.8 when they were induced for two hours by the addition of IPTG to 0.5mM. Following induction, the cells were harvested and stored at -20°C until required. The cells in the pellet from a 1 litre culture were resuspended in 5ml of 50mM Tris-HCl (pH7.5), 10% glycerol, 2mM MgCl₂, ImM DTT. Lysozyme was added to a concentration of O.lmg/ml and the cells incubated for five minutes at room temperature. They were then lysed by the addition of 10ml of detergent buffer containing 25mM Tris-HCl (pH7.5), 4mM EDTA, 0.2M NaCl, 1% deoxycholate, 1% NP40, ImM DTT and incubated at room temperature for a further 15 minutes. MgCL was added to a final concentration of lOmM with lOOμl of a 2000 units/ml DNasel solution. The extract was pipetted up and down a few times until the viscosity decreased and was then left at room temperature for 10 minutes The whole cell extract was then centnfuged at 20,000g for 30 minutes The pellet was washed three times in 0 5% NP40 (v/v), ImM EDTA and followed by one wash in 6M urea before being finally being resuspended m 1ml of 25mM Tns-HCl (pH7 5), 6M guamdme hydrochloπde, 5mM DTT After centπfugation at 13,000g for 10 minutes, the supernatant was diluted one hundred-fold mto 25mM Tns-HCl (pH7 5), 8M urea, 5mM DTT, 10% glycerol buffer and loaded onto a 2ml fast flow CM Sepharose column (Sigma) pre-equihbrated with the same buffer supplemented with 50mM NaCl

Under these conditions, denatured mariner transposase bound to the column Protein was renarured on the column by passing a 200ml linear gradient of 8-0M urea at a rate of 1 ml/mm Following renaturation, bound protein was eluted with a 20ml linear NaCl gradient of 50mM-l 0M in buffer A (20mM Tns-HCl pH7 5, ImM DTT, 10% glycerol) Fractions contaimng mariner transposase were identified by SDS-PAGE and further concentrated by spinning through a Centncon (Amicon) column (30K molecular weight cut-off) The protein was stored frozen at a concentration of about 0 25-0 5mg/ml

Example 2 Injection of manner into chicken embryos

Plasmid Mosl contaimng the mariner element (Medhora et al Genetics 128 311-318 (1991)) w as injected mto chicken embryos as descπbed by Sang & Perry (Mol Reprod Dev 1 98-106 (1989)) at a concentration of 25μg/ml together with puπfied mariner transposase at a concentration of 0 05-0 005mg/ml m a buffer contaimng lOOmM NaCl, 25mM HEPES pH7 7, 2mM dithiothreitol, 5% (v/v) glycerol, 25μg/ml bovine serum albumin, with or without 5mM Manganese acetate The injected embryos were cultured as descπbed by Perry (Nature 331 70-72 (1988)) Hatched chicks which had cells contaimng mariner sequences were identified by carrying out polymerase chain reactions (PCR) with primers specific to mariner and DNA prepared from the chonoallantoic membrane of the chicks at hatch Embryos which died during the experiments but that survived for at least 12 days after injection were also analysed for the presence of mariner sequences.

Examples 3 to 8: Materials and Methods: Plasmid Constructs and Preparation of Transposase protein

The plasmid pMoslTet was constructed by insertion of the tetracyclin resistance gene into the unique Sail site present in the open reading frame of mariner in Mosl. The Tet gene was obtained by digestion of pBR322 with Aval and EcoRI, pMosl was linearised with Sail and the two fragments ligated after treatment with Klenow polymerase to fill in the ends. The expression and preparation of recombinant-derived mariner transposase will be described in detail elsewhere (A. Dawson and D. Finnegan, in preparation). Briefly: the mariner transposase gene from pMosl was inserted into the expression vector pBCP368 (Velterop et al Gene 153 63-65 (1995)) to generate the construct pBCPMosl . This construct was transferred into E. coli strain DH5α and the cells harvested after induction of protein expression. The transposase protein was recovered as an insoluble precipitate, solubilised and bound to fast flow CM sepharose column (Sigma). The protein was renatured in 8M urea and the activity measured in an in vitro transposition assay.

Microinjection and Chick Embryo Culture

Chick embryo culture was essentially as described (Perry Nature 331 70-72 (1988)) with modifications noted in (Love et al Bio/Technology 12 60-63 (1994)). Between 1 and 2nL of uncut plasmid, at a concentration of 25μg/ml, was injected into the germinal disc of zygotes following established procedures (Love et al Bio/Technology 12 60-63 (1994)). The DNA was diluted in transposition buffer (lOOmM NaCl, 25mM HEPES pH7.9, 2mM dithiothreitol, 50mM manganese acetate, 25μg/ml BSA, 5% glycerol) and transposase protein added when required to a concentration of 15ng/ml. PCR Analysis

Tissue samples (chorioallantoic membrane, liver and gonads) were dissected from embryos which died in culture after more than 12 days of incubation and DNA extracted using Puregene (Flowgen) genomic DNA purification kit. Genomic DNA samples were obtained from chorioallantoic membrane at hatch of surviving chicks, blood samples from older birds and semen from the mature cockerel. PCR analysis was carried out on 0.5-lμg DNA samples for the presence of the mariner element and pBluescript (pMosl experiments) or for Tet^R gene and the vector chloramphenicol (CAT) resistance gene (pMoslTet experiments). Control PCR reactions, to estimate copy number, were carried out in parallel on lμg aliquots of chicken genomic DNA with pMosl or pMoslTet DNA added in quantities equivalent to that of a single copy gene (IX) a 10-fold dilution (0.1X) and a 100- fold dilution (0.01X) as described previously (Love et al Bio/Technology 12 60-63 (1994)). The primers used were:

(i) mariner

+ 5'-TCAGAAGGTCGGTAGATGGG 5'-AAATGACACCGCTCTGATCC

(ii) pBluescript

+ 5'-GCAGAGCGAGGTATGTAGGC

5'-AGCCCTCCCGTATCGTAGTT

(ni) Tet^R + 5'-CTTGAGAGCCTTCAACCCAG

5'-TTTGCGCATTCACAGTTCTC

(iv) CAT

+ 5'-AAAATGAGACGTTGATCGGC - 5'-AGGTTTTCACCGTAACACGC PCR products were analysed on 1 5% agarose gels and the copy number of the construct sequences estimated by compaπson with the control reactions

Southern Transfer Analysis and Isolation of Integrated Mariner Element DNA from Gi chicks, identified as transgenic by PCR, was digested with BamHI plus Hmdlll and EcoRI, electrophoresed through 1 % agarose gel and transferred to HybondN (Amersham) A manner-specific probe was generated by PCR using pπmers close to the ends of the element and labelled by random-pnmmg (Redipnme, Amersham) A 0 lμg aliquot of EcoRI digested DNA from Gichick 13 was ligated to lμg of Lambda Zap II EcoRI-cut arms (Stratagene) and packaged ith Gigapack Gold (Stratagene) Approximately 250,000 plaques were plated and screened with a manner-specific probe One positive plaque was identified, puπfied and the insert rescued as a plasmid following the Stratagene protocol This clone, pZapl3, was digested with EcoRI, the insert size compared to the /?. αrz ..er-hybndismg fragments present in chick 13 genomic DNA and found to co- migrate with the approximately 8kb fragment The pZapl3 clone was sequenced using pnmers near the 5' and 3' end of mariner, designed to sequence across the ends of the element mto the flanking genomic DNA

left-end pπmer 5'-TCGGCACGAAACTCGACATG πght-end pnmer 5'-GCAAATACTTAGAATAAATG

Example 3 Analysis of Chick Embryos after l ection of mariner plasmid

A seπes of expenments were earned out m which a plasmid carrying the active mariner element Mos 1 (Figure 1 (a)) (Medhora et al Genetics 128 311-318 (1991)) was injected mto chick zygotes using established procedures Puπfied marine} transposase protein was included m approximately half of the injections A total of 97 zygotes were injected, 51 w ith plasmid plus transposase protein DNA was extracted from tissues from embryos that survived for a least 12 days of incubation but died before hatch, and from the chonoallantoic membrane of hatched chicks These DNA samples were analysed by PCR to detect simultaneously the mariner element (MAR) and the plasmid vector (PBS, Figure 1(a)) The copy number of mariner and plasmid vector sequences were estimated with respect to the amount of chicken genomic DNA present single copy (one copy or more per genome equivalent), 0 05-0 5 copies per genome and less than 0 05 copies per genome 44 of the manipulated embryos survived for at least 12 days of incubation, 23 after injection of pMosl plus transposase protein

The results of the PCR analysis for the presence of mariner and plasmid vector, after injection of pMosl with and without transposase protein, are shown m Table

1 These results are compared graphically to results from injection of a standard gene construct deπved from the lysozyme gene (A Sherman, unpublished results) m Figure IB The frequency at which "single-copy" embryos were found after injection of pMosl was dramatically higher than in a lysozyme construct expenment Less than 1% of the embryos from the lysozyme expenment contained the construct at a level equivalent to one copy per genome in construct to

27% of the embryos injected with pMosl The results from analysis of embryos injected with or without transposase are very similar (Table 1, Figure 1(b)) This indicates that, if the mariner sequences detected the PCR analysis are present as a result of transposition, then transposition must be able to take place in the absence of exogenous transposase protein

The embryo samples were analysed for presence of the plasmid vector of pMosl Of the 29 embryos analysed after injection with pMosl, with or without transposase protein, which contained mariner at a lev el estimated as above 0 05 copies per genome, 6 (21%) also contained plasmid vector sequences (Table 1) The lack of detectable plasmid v ector sequences m the remaining embryos m which mariner was present, suggested that the mariner element in pMosl had transposed out of the plasmid construct, potentially mto the chicken genomic DNA Example 4 Germlme transmission of manner

Three chicks from the above expenment survived to sexual matuπty One chick was identified at hatch as potentially transgenic for pMosl Both mariner and plasmid vector sequences were detected by PCR DNA from chonoallantoic membrane and from blood samples, with a copy number of between 0 1 and 0 5 genome equivalents This estimate was confirmed when the cockerel reached sexual matunty by analysis of semen samples This cockerel was crossed with stock hens and hatched offspnng screened to detect transgenic chicks A total of 93 Gi chicks were screened, 27 (29%) of which were identified by PCR as transgenic for war er

To determine the frequency of mariner insertions m the germlme of the Go cockerel and the number of different transposition events that had occurred, individual Gi chicks were analysed Genomic DNA samples from 23 chicks were analysed by Southern blotting to identify the number and size of restnction fragments that contained insertions of mariner The genomic DNA samples were digested with restnction enzymes BamHI and Hindlll, that do not cut withm mariner itself (Figure 1(a)), and hybndised with a mariner-specific probe (Figure 2(a)) Analysis using a further restnction enzyme, EcoRI (Figure 2(b)), that also does not cut withm mariner, enabled us to determine the number of manner- hybndismg restnction fragments present in the different Gi chicks Each Gi chick was classified by the size of BamHI/Hindlll and EcoRI, mαrmer-hybndismg restnction fragments (Table 2) Six different fragments containing mariner were identified and an example of each is shown in Figures 2(a) and (b) Three fragments (Table 2, Figures 2(a) and (b), lane 7), were the most common and were clearly present in the parent cockerel (Figures 2(a) and (b), lane 8) Three of the Gi chicks were identified by PCR as containing the plasmid vector sequences, as well as mariner Southern transfer analysis (e g Figures 2(a) and (b), lane 1) indicated that they contained a 5kb (BamHI/Hmdlll digest) or an 8kb (EcoRI digest) restnction fragment This observation indicates that these transgenic birds resulted from integration of multiple copies of the intact pMosl plasmid, which explains the detection by PCR of plasmid vector sequence in genomic DNA samples from the Go cockerel. The analysis of genomic DNA from Gi chicks suggested that mariner had transposed from pMosl into the chromosomes of the Go cockerel at an early stage of development, and that multiple transposition events had taken place.

Example 5: Transposition of mariner into the chicken genome

To demonstrate that the restriction fragments hybridising to mariner do correspond to copies of mariner integrated into chicken genomic DNA, a restriction fragment containing a single copy of mariner was cloned from genomic DNA of one Gi chick (chick 13; Figure 2(b), lane 2). A library of EcoRI fragments from chick 13 was constructed and screened with a mariner probe. A clone containing an 8.2kb fragment, corresponding to the lower band in Figure 2(b) lane 2, was isolated. This clone, pZAP13, was used to reprobe the Southern blot shown in Figure 2(b), (Figure 3(a)). The probe identified a series of EcoRI restriction fragments in all the chicken genomic DNA samples, including DNA from a wild-type chick (Figure 3(a), lane 9). The mariner hybridising fragments are also faintly detectable. The clone was also analysed by DNA sequencing, using pnmers internal to the ends of mariner, designed to prime sequence over the ends of the inserted mariner element, if complete. The sequence generated (Figure 3(b)) corresponds exactly to the sequence of the ends of the mariner element but is flanked by sequences that differ from the Drosophila genomic DNA adjacent to the element in pMosl. The element present in the chicken DNA is flanked by TA dinucleotide repeats, the sequence^' characteristically generated by mariner transposase-mediated transposition. These results indicate that mariner had integrated into chicken chromosomal DNA by transposition from pMosl .

Example 6: Source of Transposase Function

There was no evidence that the inclusion of transposase protein with the pMosl plasmid in the zygote injection experiments was necessary for transposition (Figure

1(b) and Table 1). The transposase activity could have been due to expression of the Mosl transposase gene or an endogenous activity present in the chick zygote. In order to distinguish between these possibilities, a series of zygote injection experiments was carried out using a construct in which the mariner transposase gene was inactivated by insertion of the tetracyclin resistance gene (Tet^R) within the transposase coding region (pMoslTet). Transposase protein was again included in approximately half of the zygote injections. DNA samples from embryos and chicks were analysed by PCR for the presence of the Tet^R gene and the plasmid vector and their copy number estimated. 29 embryos injected with plasmid alone and 34 with plasmid plus exogenous transposase were analysed and the results are shown graphically in Figure 4. The proportion of embryos containing the Tet^R sequence at a level above 0.05 genome equivalents (17% of embryos without transposase and 24% of embryos with transposase) is much lower than detected after introduction of intact mariner (66% of embryos without transposase and 78% of embryos with transposase). All of the embryo DNA samples that contained mariner sequences also contained detectable amounts of the plasmid vector (data not shown). The small number of embryos that contained the construct at a single level could have been the result of random integration of the intact plasmid. Again, there was no evidence for function of the exogenous transposase. These results suggest that the transposase activity that lead to transposition of the mariner element from pMosl into the chicken genomic DNA was derived from expression of functional transposase by the construct. The results do not exclude the possibility that the exogenous transposase protein was functional, but they do indicate that it was not necessary for transposition of mariner from pMosL

Example 7: Germline Stability of Integrated Mariner Elements Two Gi birds, cockerels 3 and 7, that each had a single copy of mariner integrated at different chromosomal sites (Figures 2(a), lanes 4 and 5) were selected to analyse stability of the elements after germline transmission to the G₂ generation. They were each crossed with stock hens, DNA extracted from resulting embryos and screened by PCR to identify transgenic embryos. The ratios of transgenic to non-transgemc offspnng from cockerel 3 (65 59) and cockerel 7 (64 57) did not differ significantly from the expected 1 1 Mendehan ratio The genomic DNA from transgenic embryos was digested with BamHI and Hmdlll and the pattern of mariner-hybndismg fragments compared to the single band present in the transgenic parent All of the transgenic offspnng from both cockerels had a single mariner band that co-migrated with the restnction fragment present in the parent cockerel (data not shown) There is no evidence of instability of mariner after transposition, although a low level of instability would not have been detected

Example 8 Testing mariner transposition in mice

An intact plasmid construct (pMosl), will be injected mto the pronucleus or the cytoplasm of mouse fertilised eggs at a concentration of approximately 1 5ng/μl The method used is as descnbed by Whitelaw et al (Biochemical J 286 31-39 (1992)) and is based on the work of Bnnster et al (P oc Nat'l Acad Sci USA 82 4438-4442 (1985)) In some expenments recombmant-denved, puπfied marine! transposase protein or mRNA. will be included The mouse embryos will be transferred to surrogate mothers and new-born mice will be screened to identify any transgenic for mariner All transgenic mice will be analysed further to determine if the manner element is present in the mice as a result of transposase- catalysed transposition or random integration of the whole plasmid construct

Discussion

The present studies show that the Diosophila mauntiana transposable element marine! can transpose mto the chicken genome after its introduction into the chick zygote by micro injection The fate of a manner -contaimng plasmid was analysed after injection into chick embryos at the zygote state and embryo development of at least 12 days In contrast to the previous results, following the same procedures but using a vanety of gene constructs, it was found that mariner was present at a level equiv alent to one copy per cell in over 20% of embryos The plasmid vector that earned the mariner element was not detectably present in almost 80% of these embryos These results suggested that the mariner element transposed out of the ongmal plasmid and had integrated mto the chicken genome This interpretation was confirmed by analysis of a cockerel transgenic for mariner that survived to sexual matunty and transmitted copies of mariner to nearly 30% of his offspnng Analysis of individual Gi birds showed that a total of six different insertions of mariner were present in different individuals Furthermore, isolation of a single copy of mariner from a Gi transgenic bird confirmed that a complete element had transposed mto chicken genomic DNA and that the transposition event had generated the expected TA repeat at the insertion site (Bryan et al Genetics 125 103-1 14 (1990)) No evidence for stability of integrated copies of mariner was obtained after germlme transmission to the G₂ generation

The frequency of mariner transposition mto the chicken genome indicated by this analysis is high (over 20%), although this has to be confirmed by the generation of additional transgenic birds The frequency of germlme transformation obtained by introduction of the same marine! element mto Drosophila melanogaster vaned between 4 and 31% (Garza et al Genetics 128 303-310 (1991)), a comparable frequency The proportion of Gi birds that inhented manner from the Go cockerel was approximately 30%, a 10-fold higher transmission frequency than obtained after introduction of linear gene constructs (Love et al Bio/Technology 12 60-63 (1994)) The analysis of Gi birds indicated that there had been multiple insertions of mariner Two possible explanations cannot yet be distinguished between either that several independent transposition events took place from the introduced plasmid or that a copy of mariner transposed into the chicken genome and that this was followed by secondary transposition events The fact that two copies of mariner w ere stably transmitted to the G₂ generation suggests that, once integrated mto the chicken genome, mariner elements are stable Mariner is also stable after transposition mto the D melanogaster genome with an excision rate estimated as below 0 1% (Lohe et al Genetics 140 183-192 (1995)) Punfied transposase protein was included with pMosl DNA in half of the micromjections but the PCR analysis suggested that the frequency of transposition was not increased by the addition of the enzyme No transposition was detected when the transposase gene was inactivated by insertion of the Tet^R It is therefore concluded that the mariner transposition events observed were catalysed bv expression of the transposase gene in pMosl Previously it has been shown that plasmid DNA injected mto the chick zygote is replicated approximately 20-fold dunng the first 24 hours of embryo development (Sang, H M , and Perry, M M Mol Reprod Dev 1 98-106 (1989)) and that expression of a reporter gene construct can be detected withm 9 hours of injection (Perry et al Roux's Arch De\ Biol 200 312-319 (1991)) It is therefore predicted that there will be a high copv number of pMosl per cell dunng the early stages of dev elopment that can act as template for transcnption of the transposase gene Even if the transcπption and translation is ineffective sufficient transposase may be synthesised to catalyse transposition Once the mariner elements have integrated they are apparently stable, even though they carry an intact transposase gene Expression of the transposase gene may be very inefficient once the elements are incorporated in chicken chromosomes and there will only be a small number of copies per cell

These results, and recently descnbed evidence for transposition of mariner m zebrafish (Fadool et al Proc Nat'l Acad Sci US 4 95 5182-5186 (1998)), are encouraging evidence to support the development of mariner as a vector for transgenesis for vertebrates The use of a manner-denv ed vector for transgenesis has several potential advantages, particularly for transgenic manipulation of poultry The frequency of integration may be increased above the level currently possible The observation that multiple integration ev ents are present in the germlme of one Go transgenic bird suggests that several transgenic lines, with insertions at different genomic sites, may be established by breeding from one founder bird There is accumulating evidence that expression of single copy transgenes is less subject to down-regulation of expression than transgenes integrated in multicopy arrays (Garπck et al Nature Genetics 18 56-59 (1998)) The fact that mariner vectors will integrate transgenes as single copies may have the additional advantage of resulting in more predictable levels of transgene expression. It is planned to investigate methods to provide the transposase activity in trans to a mariner vector carrying a transgene. The frequency of transposition of a mariner element modified to incorporate a transgene and the size of transgene a mariner vector can carry have to be established. Analysis of transgene expression will establish if expression of transgenes introduced in mariner vectors as single copies is more predictable than expression of transgenes in multicopy arrays. Mariner is only one of the superfamily of mαrmer-like-elements which have potential for development of vectors for transgenesis (Dawson, A. and Finnegan, D. J., Nature Bio. 16 20-21 (1998); Raz et al Current Biol. 8 82-88 (1998)). Future developments will demonstrate if particular elements are more effective as vectors than others or if specific elements are more useful for one application than another.

TABLE 1

PCR analysis of DNA from embryos and chicks that developed after injection of pMosl, with or without addition of recombinant-derived transposase protein

ro

* = Estimation of plasmid copy number is explained in Materials and Methods a = PCR using primers specific sequence b = PCR using primers specific to plasmid vector sequences

TABLE 2

Estimated size of «/«r //er-hybridising restriction fragments and their frequency in Gi transgenic birds.

co

Claims

1 A method for the preparation of a transgenic ammal embryo comp╧Çsing the step of introducing a manner-hke element (MLE) containing a transgene mto an animal embryo cell, optionally including the step of introduction of exogenous transposase protein, or a DNA or RNA sequence encoding a transposase

2 A method as claimed m claim 1 , m which the ammal is an avian species

3 A method as claimed in claim 2, m which the animal is a poultry species, for example Gal I us domesticus

4 A method as claimed in claim 1, m which the animal is an ungulate species

5 A method as claimed in claim 3, in which the animal is a cow or a bull, sheep, goat, water buffalo, camel or pig

6 A method as claimed m any one of claims 1 to 5, m which the manner-like element is the transposable element mariner from Di osoplula mauritiana

7 A method as claimed in any one of claims 1 to 6, in which the manner-hke element contaimng the transgene is introduced into the cell in a construct

8 A method as claimed m any one of the preceding claims, m which the introduction of the manner-hke element (MLE) is by injection of the MLE mto the cytoplasm or mto the pronucleus of a zygote or the nucleus of an animal embryo cell, by electroporation or using hposomes

9 A method for the preparation of a transgenic ammal embryo comp╧Çsing the step of introducing a manner-hke element (MLE) containing a transgene into an ammal adult cell, optionally including co-mtroduction of exogenous transposase protem, or a DNA or RNA sequence encoding a transposase

10 A method for the preparation of a transgenic ammal embryo comp╧Çsing the step of introducing a manner-hke element (MLE) contaimng a transgene mto an ammal foetal cell, optionally including co-mtroduction of exogenous transposase protein, or a DNA or RNA sequence encoding a transposase

11 A method as claimed m any preceding claim, in which the resulting ammal embryo is prepared by (l) removmg the nucleus of the cell followmg introduction of the manner- ke element contaimng the transgene and its insertion mto the chromosomes, and (n) subsequently introducing the nucleus mto an enucleated oocyte which is allowed to develop mto an ammal embryo

12 A method for the preparation of an ammal, the method comp╧Çsing the steps of

(a) prepanng an embryo according to any one of the preceding claims,

(b) causing an animal to develop to term from the embryo, and

(c) optionally breeding from the ammal so formed

13 A method as claimed m claim 12, m which the ammal embryo is further manipulated pnor to full development of the embryo

14 A transgenic ammal prepared by a method according to claim 12 or claim 13

15 The use of a manner- ke element m the therapy of a disease condition caused by the absence of a gene or the mutation of a gene

16 The use of a manner-hke element in the preparation of an agent for the prophylaxis or treatment of a disease caused by the absence of a gene or the mutation of a gene

17. A method of treatment of a disease condition caused by the absence of a gene or the mutation of a gene, comprising the step of introducing a mariner-like element containing a transgene into an animal cell, optionally including introduction or transposase protein, or a DNA or RNA sequence encoding transposase.