CA2300972A1

CA2300972A1 - Use of mariner transposan in the production of transgenic animals

Info

Publication number: CA2300972A1
Application number: CA002300972A
Authority: CA
Inventors: Helen Sang; David John Finnegan
Original assignee: Individual
Current assignee: Roslin Institute Edinburgh
Priority date: 1997-08-22
Filing date: 1998-08-21
Publication date: 1999-03-04
Also published as: JP2001513336A; EP1006790A1; AU8817798A; WO1999009817A1

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.
S 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 Did a 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 218 348-3S3 (1982) and US-A-.670388).
2S A method for culturing the chick embryo from the newly 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 cyrtoplasm 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 Roux'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 BiolTechnology 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 clucks hatched after DNA injection have incorporated the injected DNA into their 10 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 D:~tA 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.

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 maa~ritiana but closely related elements have been discovered in a wide variety of species both vertebrate and invertebrate (Robertson, H. M., Nata~re WO 99/0981'7 PCT/GB98/02517 ( 1993)). It has also been used to investigate pathogenic organisms such as Leishmanin (Gueiros-Filho, F. J., & Beverley, S. M., Science 276 1716-1719 (1997) and Hartl, D. L., Science 276 1659-1660 (1997). However, the use of mariner described by Gueiros-Filho et al and Hartl only relate to its use as a genetic tool, i.e.
5 for insertional mutagenesis and not as a means for the preparation of transgenic animals by transgenesis or mutagenesis. In summary, mariner has not been described as being suitable for a role in the production of transgenic animals and nor is such a use contemplated in the prior art.
10 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 comprising the step of introducing a 15 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 term transgenic is used in the context of the present invention to describe animals 20 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 25 proportion such that its progeny stably inherit the transgene. Where the transgene is only present in a proportion of cells of the atumal, 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 in their somatic cells without passing the gene onto their offspring.

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 5 has been the subject of the introduction of a mariner-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 15 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 Gallirs domesticirs, 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 trans~ene into an animal embryo cell. The embryo cell may be at the 25 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 mariner-like element may be the transposable element mariner from Drosophila maaeritiana or a closely related element from another vertebrate or invertebrate species (Robertson, H. M., Nature 362 241 (1993)). The mariner-like element may conveniently be derived from the cells of the animal whose chromosomes are to 5 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.
ll~lariner-like transposable elements are about 1,300bp long with terminal inverted repeats of about 30bp. Each mariner-like element encodes a polypeptide that is a 10 putative transposase and that has, on average, 34°,io amino acid sequence identity with the polypeptides encoded by other mariner-like elements. The amino acid sequences of the putative transposases of all mariner-like elements include a characteristic motif known as D, D34D, where "D" represents an aspartate residue. The third aspartate of this motif is followed immediately by a tyrosine residue (Robertson, H. M., J.
Insect 15 Physiol. 41 99-105 (1995)).
The transgene can be contained in the mariner-like element at any point within the mariner sequence. Without being bound by theory, it is believed that approximately the final 100 bases of each end of the mariner--like element may be important for 20 function of the MLE and its incorporation into the chromosomes of a cell.
Thus the transgene may be positioned anywhere within 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.
25 The transgene sequence contained in the mariner-like element may be any desired forei5n 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, myoglobin, cytochromes, etc. Other gene sequences 30 may encode proteins whose genes are absent or mutated such that the corresponding protein is not produced or is not produced in active form, i.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 animal may be used in Xenotransplantion into a recipient which is allogenic for the immune proteins being expressed in the cells. Similarly, allotransplantation is also included.
In agricultural applications of the method of the present invention may be used to produce improved farm animals. The trans~enes introduced into the animals may include, but are not limited to, disease resistance genes, growth enhancing genes or 1 S genes which provide for improved characteristics in a particular trait or introduction of a novel trait.
The introduction of the mariner-like element (MLE) may conveniently be achieved by injection of the MLE into the cytoplasm or into the pronucleus of a zygote or the 20 nucleus of an animal embryo cell. Other routes of introduction, such as electroporation or liposomes may be equally effective and used in the method according to the present invention.
The MLE may be introduced in the form of a construct comprising the DNA
25 sequence of a mariner-like element and the desired transgene or simply the nucleotide sequence of a mariner-like 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 Bacterial Artificial Chromosome (BAC). The constructs 30 may also contain additional regulatory sequences, if required, such as promoters or enhancers, depending of the foreign DNA being introduced. A further aspect of the present invention is therefore a construct or a vector comprising a mariner-like element containing a transgene as described above. In general, the MLE will be cloned in a plasmid vector for ease of manipulation and cloning-in 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 mariner-like element, or shortly before or shortly thereafter, the method of the present invention may contain an additional optional step of introducing exogenous transposase protein, or a DNA or RNA sequence encoding I 0 a transposase.
The mariner-like element and the transposase introduced into the cell may be derived from the same animal species or different species.
I 5 According to a second aspect of the present invention there is provided 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 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 comprising the step of introducing a mariner-like element (MLE) containing a transgene into an animal foetal cell.
optionally including the step of introduction of exogenous transposase protein, or a DNA or R~'~1A sequence encoding a transposase.
In any one of the methods according to the first, second and third aspects of the present invention, the resulting animal embryo may be prepared by (i) removing the nucleus of the cell following introduction of the mariner-like element containing the 30 transgene and its insertion into the chromosomes, and (ii) subsequently introducing the nucleus into an enucleated oocyte which is allowed to develop into an animal g 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 Vl~'O-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 Natatre 380 64-66 (1996); or Cibelli et al Science 280 1256-10 (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 Theriogenologv 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-942427-1.
WO-A-9807841, WO-A-9827214, WO-A-900343, 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 (BLWF1) and an 25 epithelial-like cell line derived from a 9-day old sheep embryo (SEC 1 ) 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 96/0773?.
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 (Nattcre Nledicine 4 (5) 569-574 (1998) and in Cibelli et al (Science 280 1256-( 1998)).
Where the donor cells are described 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 5 described in WO-A-9707669. The mitotic cell cycle has four distinct phases, G1, S.
G2 and M. The beginning event in the cell cycle, called start, takes place in the G1 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 G1 phase, which is the pre-DNA synthesis phase. The second stage, 10 the S phase, is when DNA synthesis takes place. This is followed by the G2 phase.
which is the period 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 in any of these four phases of the cycle; they 15 are usually described as being 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 20 including chemical treatments, nutrient deprivation, growth inhibition or manipulation of gene expression. Presently the reduction of serum levels in the culture medium has been used successfully to induce quiescence in both ovine and bovine cell lines. In this situation, the cells exit the growth cycle during the G1 phase and arrest, as explained above, in the so-called GO stage. Such cells can remain in 25 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 point in 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 centrioles, reduction or 30 complete cessation in all protein synthesis, increased proteolysis, decrease in transcription and increased turnover of RNA resulting in a reduction in total cell RNA, disaggregation of polyribosomes, accumulation of inactive 80S ribosomes and chromatin condensation (reviewed Whitfield et al., Control of Animal Cell Proliferation, l 331-365 (1985)).
5 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 10 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.
15 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 favaur the use of cleavage-stage embryos in some species is the extent to which reprogramming of gene expression is required.
25 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 (Howiett & Bolton .l.
Ernbryol.
E.rp. Moyhol. 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 experimental confirmation of this judgement. The original procedure described for ungulates involved splitting the cell into two halves, one of which was 5 likely to be enucleated {Willadsen Natacre 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 believed to accelerate the pattern of differentiation of the new embryo {Eviskov et ul., 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 in 67%
of sheep oocvtes (Smith & Wilmut Biol. Reprorl. 40 1027-103 (1989)). Only with the 15 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., .l. Reprod. Fertil. 82 173 ( 1988)). In livestock species, this is probably the method of routine use at present (Prather & First J. Reprod.
Fertil.
Sccppl. 41 125 (1990), Westhusin et al., Biol. Reprod. (Suppl.~ 42 176 (1990)).
There ha~~e been very few reports of non-invasive approaches to enucleation in mammals, whereas in amphibians, irradiation with ultraviolet light is used as a routine procedure (Gurdon Q. J. tLlicrosc. Soc. 101 299-311 (1960)). There are no detailed reports of the use of this approach in mammals, although during the use of 25 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 animal, the method comprising the steps of:

(a) preparing an embryo according to any of the preceding aspects of the presentinvention;
(b) causing an animal to develop to terns from the embryo; and (c) optionally breeding from the animal so formed.
The animal embryo prepared in accordance with this aspect of the present invention may be further manipulated prior to full development of the embryo. This may include the introduction of additional genetic material or to assay the embryo for particular genetic characteristics or the presence or absence of a gene. It is also possible that more than one animal can be derived 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 animal 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 mariner-like element in 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 mariner-like element in the preparation of an agent for the prophyiaxis or treatment of a disease caused by the absence of a gene or the mutation of a gene. Such methods of treatment may comprise the introduction of a mariner-like element containing a transgene into an animal 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 in which the cells of the embn.~o 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 n:artatis nu~tandis.
5 The present invention will now be described 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 in 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 mariner-containing plasmid pMosl. FIGURE 1(a) shows a diagram of p:~losl, indicating sequences identified by PCR and unique restriction sites.
FIGURE 1 (b) shows a graphical presentation of the results given in Table 15 1. The estimated copy number per genome equivalent of a lysozyme gene construct, injected in a previous series of experiments (n=18b), is compared to the estimated copy number of mariner sequence. The results of injecting ptLlosl with and without the addition of recombinant-derived transposase protein are compared.
FIGURE 2 shows a Southern blot analysis of Uenomic DNA isolated from individual G, transgenic chicks, hybridised with a mariner probe. FIGURE
2(a) shows BamHI/HindIII digests of individual chicks which each have a novel pattern of mariner-hybridising fragments ( lanes 1 to 7), and of the 25 parent Go cockerel (lane 8). A control digest of non-transgenic 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 pZAPl3 (see FIGURE 3).
FIGURE 3 shows the characterisation of a single integrated mariner element. FIGURE 3(a) shows the Southern blot of genomic DNA from individual G, chicks digested with BamHI and HindIII (from FIGURE
2(a)) was stripped and reprobed with the EcoRI insert from pZAP 13.
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 pZAPl3.
FIGURE 4 shows PCR analysis for present of the TetR gene in DNA from embryos and chicks that survived for at least 12 days of incubation after 10 introduction of pMos 1 Tet. The copy number of the TetR 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 15 The mariner transposase used in the following experiments was purified from E. coli strain BL21 DE3 (Studier et al Methods in En~ymolow 185 60-89 (1991)) carrying the plasmid pBCPMosI. 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 tLlosl was inserted at the lfdeI site of pBCPMosl.
20 These cells were grown in Luria broth (LB) in an orbital shaker (204rpm, 37°C} to an ODS;o of 0.8 when they were induced for two hours by the addition of IPTG to O.~mM. 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 Sml of SOmM Tris-HCl (pH7.5), 10% glycerol, 2mM MgCI~, lm:'~I DTT. Lysozyrne was 25 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 lOml of detergent buffer containing 25mM Tris-HCl (pH7.5), 4mM EDTA, 0.2M NaCI, 1% deoxycholate, 1%
NP40, 1mM DTT and incubated at room temperature for a further 15 minutes.
MgCh was added to a final concentration of lOmM with 100p1 of a 2000 units/ml 30 DNaseI 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 centrifuged at 20,OOOg for 30 minutes. The pellet was washed three times in 0.5% NP40 (v/v), 1mM EDTA and followed by one wash in 6M urea before being finally being resuspended in lml of 25mM Tris-HCl (pH7.5), 6M
5 guanidine hydrochloride, SmM DTT. After centrifugation at 13,OOOg for 10 minutes, the supernatant was diluted one hundred-fold into 25mM Tris-HCl (pH7.5), 8M
urea, ~mM DTT, 10% glycerol buffer and loaded onto a 2m1 fast flow CM Sepharose column (Sigma) pre-equilibrated with the same buffer supplemented with SOmNi NaCI.
Under these conditions, denatured mariner transposase bound to the column.
Protein was renatured on the column by passing a 200m1 linear gradient of 8-OM urea at a rate of lm>r~min. Following renaturation, bound protein was eluted with a 20m1 linear NaCI gradient of ~OmM-1.OM in buffer A (20mM Tris-HCl pH7.5, 1mM DTT, 10%
15 glycerol). Fractions containing mariner transposase were identified by SDS-PAGE
and further concentrated by spinning through a Centricon (Amicon) column (34K
molecular weight cut-offJ. The protein was stored frozen at a concentration of about 0.25-O.Smg/ml.
Example 2: Injection of mariner into chicken embryos Plasmid Rlosl containing the mariner element (Medhora et al Genetics 128 311-(1991)) was injected into chicken embryos as described by Sang & Peny (Mol.
Reprod. Dev. 1 98-106 (1989)) at a concentration of 2~pg/ml together with purified mariner fransposase at a concentration of 0.05-O.OO~mgiml in a buffer containing 25 100mM NaCI, 2~mM HEPES pH7.7, 2mM dithiothreitol, 5% (v/v) glycerol, 2~pg/ml bovine serum albumin, with or without SmM Manganese acetate. The injected embryos were cultured as described by Perry (Nata~re 331 70-72 (1988)).
Hatched chicks which had cells containing mariner sequences were identified by carrying out polymerase chain reactions (PCR) with primers specific to mariner and DNA
prepared from the chorioallantoic 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 S Plasmid Constructs and Preparation of Transposase protein The plasmid pMoslTet was constructed by insertion of the tetracyclin resistance gene into the unique SaII site present in the open reading frame of mariner in pMosl. The TetR gene was obtained by digestion of pBR322 with AvaI and EcoRI, p~Llosl was linearised with SaII and the two fragments ligated after treatment with Klenow polymerise 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 pBCPMosI.
This 1 S construct was transferred into E. coli strain DH~a 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 BiolTechnology 12 60-63 (1994)).
Between l and 2nL of uncut plasmid, at a concentration of 25p.g/ml, was injected into the germinal disc of zygotes following established procedures (Love et al BiolTechnology 12 60-63 (1994)). The DNA was diluted in transposition buffer (100mM NaCI, 25mM HEPES pH7.9, 2mM dithiothreitol, SOmM manganese acetate, 2~ug/ml BSA, 5% glycerol) and transposase protein added when required to a concentration of l5ng/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 5 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-lp.g DNA samples for the presence of the mariner-element and pBluescript (pMosl experiments) or for TetR gene and the vector chloramphenicol {CAT) resistance gene (pMoslTet experiments). Control PCR
10 reactions, to estimate copy number, were carried out in parallel on leg aliquots of chicken genomic DNA with pulosl or pMoslTet DNA added in quantities equivalent to that of a single copy gene ( 1 X) a 10-fold dilution (0.1 X) and a 100-fold dilution (0.01X) as described previously (Love et al BiolTechnology 12 60-{ 1994)). The primers used were:
(i) mariner + 5'-TCAGAAGGTCGGTAGATGGG
- 5'-AAATGACACCGCTCTGATCC
(ii) pBluescript + S'-GCAGAGCGAGGTATGTAGGC
- 5'-AGCCCTCCCGTATCGTAGTT
(iii) Tet~
25 + 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 comparison with the control reactions.
Southern Transfer Analysis and Isolation of Integrated Mariner Element 5 DNA from G, chicks, identified as transgenic by PCR, was digested with BamHI
plus HindIII and EcoRI, electrophoresed through 1 % agarose gel and transferred to HybondN (Amersham). A mariner-specific probe was generated by PCR using primers close to the ends of the element and labelled by random-priming (Rediprime, Amersham). A O.lug aliquot of EcoRI digested DNA from G~chick 10 13 was ligated to 1 pg of Lambda Zap II EcoRI-cut arms (Stratagene) and packaged with Gigapack Gold (Stratagene). Approximately 250,000 plaques were plated and screened with a mariner-specific probe. One positive plaque was identified.
purified and the insert rescued as a plasmid following the Stratagene protocol.
This clone, pZapl3, was digested with EcoRI, the insert size compared to the 15 mariner-hybridising fragments present in chick 13 genomic DNA and found to co-migrate with the approximately 8kb fragment. The pZapl3 clone was sequenced using primers near the 5' and 3' end of mariner, designed to sequence across the ends of the element into the flanking genomic DNA:
20 left-end primer ~'-TCGGCACGAAACTCGACATG
right-end primer 5'-GCAAATACTTAGAATAAATG
Example 3: Analysis of Chick Embryos after injection of mariner- nlasmid A series of experiments were carried out in which a plasmid carrying the active 25 mariner element tt~losl (Figure 1(a)) (Medhora et al Genetics 12$ 311-318 (1991)) was injected into chick zygotes using established procedures. Purified mariner-transposase protein was included in approximately half of the injections. A
total of 97 zygotes were injected, 51 with plasmid plus transposase protein. DNA was extracted from tissues from embryos that survived for a least 12 days of incubation 30 but died before hatch, and from the chorioallantoic membrane of hatched chicks.

WO 99109817 PCTlGB98/02517 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 5 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 in Table 1. These results are compared graphically to results from injection of a standard gene construct derived from the lysozyme gene (A. Sherman, unpublished results) in Figure 1 B. The frequency at which "single-copy" embryos were found after injection of pMosl was dramatically higher than in a lysozyme construct 15 experiment. Less than 1 % of the embryos from the lysozyme experiment 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 in the PCR analysis are present as 20 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 25 transposase protein, which contained mariner at a level estimated as above 0.0~
copies per genome, 6 (21 %) also contained plasmid vector sequences (Table 1 ).
The lack of detectable plasmid vector sequences in the remaining embryos in which mariner was present, suggested that the mariner element in pMosl had transposed out of the plasmid construct, potentially into the chicken genomic 30 DNA.

Example 4: Germline transmission of mariner Three chicks from the above experiment survived to sexual maturity. One chick was identified at hatch as potentially transgenic for pMosl. Both mariner and plasmid vector sequences were detected by PCR in DNA from chorioallantoic 5 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 maturity by analysis of semen samples. This cockerel was crossed with stock hens and hatched offspring screened to detect transgenic chicks. A total of 93 G, chicks were screened, 27 (29%) of which were identified by PCR as 10 transgenic for mariner.
To determine the frequency of rnariner insertions in the germline of the Go cockerel and the number of different transposition events that had occurred, individual G, chicks were analysed. Genomic DNA samples from 23 chicks were 15 analysed by Southern blotting to identify the number and size of restriction fragments that contained insertions of mariner. The genomic DNA samples were digested with restriction enzymes BamHI and HindIII, that do not cut within mariner itself (Figure I(a)), and hybridised with a mariner-specific probe (Figure 2(a)). Analysis using a further restriction enzyme, EcoRI (Figure 2(b)), that also 20 does not cut within mariner', enabled us to determine the number of nrariner-hybridising restriction fragments present in the different G, chicks. Each G~
chick was classified by the size of BamHI/HindIII and EcoRI, mariner-hybridising restriction fragments (Table 2). Six different fragments containing mariner were identified and an example of each is shown in Figures 2(a) and (b). Three 25 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 G~ 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 Skb (BamHI/HindIII digest) or an 8kb (EcoRI
30 digest) restriction 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 G, 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 G~
chick (chick 13; Figure 2(b), lane 2). A library of EcoRI fragments from chick 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, pZAPl3, 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 primers 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 T.A 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 carned out using a construct in which the mariner transposase gene was inactivated by insertion of the tetracyclin resistance gene (TetR) within S 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 TetR 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 10 the results are shown graphically in Figure 4. The proportion of embryos containing the TetR sequence at a level above 0.0~ 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
15 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 20 transposition of the mariner element from p~Llosl 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 G~ 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 Gz generation.
30 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-transgenic offspring from cockerel 3 (65:59) and cockerel 7 (64:57) did not differ significantly from the expected 1:1 Mendelian ratio. The genomic DNA
from transgenic embryos was digested with BamHI and HindIII and the pattern of mariner-hybridising fragments compared to the single band present in the 5 transgenic parent. All of the transgenic offspring from both cockerels had a single mariner band that co-migrated with the restriction 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 into the pronucleus or the cytoplasm of mouse fertilised eggs at a concentration of approximately l.5ng/~1.
The method used is as described by Whitelaw et al (Bioclzemical J. 286 31-39 (1992)} and is based on the work of Brinster et al (Proc. Nat'I Acad. Sci. USA

15 4438-4442 (1985)). In some experiments recombinant-derived, purified mariner 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 mariner 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 Drosophila mauritiana transposable element mariner can transpose into the chicken genome after its introduction into the chick zygote by microinjection. The fate of a mariner-containing 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 variety of gene constructs, it was found that mariner was present at a level equivalent to one copy per cell in over 20% of embryos. The plasmid vector that carried the mariner element was not detectably present in almost 80% of these embryos. These results suggested that the mariner element transposed out of the original plasmid and had integrated into the chicken genome. This interpretation was confirmed by analysis of a cockerel transgenic for mariner that survived to 5 sexual maturity and transmitted copies of mariner to nearly 30% of his offspring, Analysis of individual G, 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 G, transgenic bird confirmed that a complete element had transposed into chicken genomic DNA and that the transposition event had 10 generated the expected TA repeat at the insertion site (Bryan et al Genetics 125 103-114 (1990)). No evidence for stability of integrated copies of mariner was obtained after germline transmission to the GZ generation.
The frequency of mariner transposition into the chicken genome indicated by this 15 analysis is high (over 20%), although this has to be confirmed by the generation of additional transgenic birds. The frequency of germline transformation obtained by introduction of the same mariner element into Drosophila melanogaster varied between 4 and 31% (Garza et al Genetics 128 303-310 (1991)), a comparable frequency. The proportion of G, birds that inherited mariner from the Go cockerel 20 was approximately 30%, a 10-fold higher transmission frequency than obtained after introduction of linear gene constructs (Love et al BiolTechnologv 12 60-{1994)). The analysis of G~ 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 25 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 were stably transmitted to the Gs generation suggests that, once integrated into the chicken genome, mariner elements are stable. tLlariner is also stable after transposition into the D. rnelanogaster genome with an excision rate estimated as 30 below 0.1 % (Lohe et al Genetics 140 I83-192 (1995)).

Purifed transposase protein was included with pMosl DNA in half of the microinjections 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 TetR. It is therefore 5 concluded that the mariner transposition events observed were catalysed by expression of the transposase gene in pMosl. Previously it has been shown that plasmid DNA injected into the chick zygote is replicated approximately 20-fold during the first 24 hours of embryo development (Sang, H. M., and Perry, M.
M., ll~lol. Reprod. Dev. 1 98-106 (1989)) and that expression of a reporter gene 10 construct can be detected within 9 hours of injection (Perry et al Rot~.r's Arch. Deu.
Biol. 200 312-319 ( 1991 )). It is therefore predicted that there will be a high copy number of pMosl per cell during the early stages of development that can act as template for transcription of the transposase gene. Even if the transcription and translation is ineffective sufficient transposase may be synthesised to catalyse 15 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.
20 These results, and recently described evidence for transposition of mariner in zebrafish (Fadool et al Proc. Nat'l. Acad. Sci. USA 95 ~ 182-5186 (1998)), are encouraging evidence to support the development of mariner as a vector for transgenesis for vertebrates. The use of a mariner-derived vector for transgenesis has several potential advantages, particularly for transgenic manipulation of 25 poultry. The frequency of integration may be increased above the level currently possible. The observation that multiple integration events are present in the germline 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 30 transgenes is less subject to down-regulation of expression than transgenes integrated in multicopy arrays (Garrick et al Naiure Generics 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 traps to a mariner vector carrying a transgene. The frequency of transposition 5 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 mariner-like-elements which 10 have potential for development of vectors for transgenesis (Dawson, A. and Finnegan, D. J., Natacr-e Bio. 16 20-21 (1998); Raz et al Current Biol. 8 82-(1998)). Future developments will demonstrate if nart1C111ar PIPmPntc arP mnra effective as vectors than others or if specific elements are more useful for one application than another.

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Claims

1. A method for the preparation of a transgenic avian or non-human mammalian embryo comprising the step of introducing a mariner-like element (MLE) containing a transgene into an avian or non-human mammalian embryo cell, optionally including the step of introduction of exogenous transposase protein or DNA encoding a transposase.

2. A method as claimed in claim 1, in which the avian is a poultry species, for example Gallus domesticus.

3. A method as claimed in claim 1, in which the non-human mammal is an ungulate species.

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

5. A method as claimed in claim 1, in which the non-human mammal is a rodent species.

6. A method as claimed in claim 5, in which the rodent is a rat or a mouse.

7. A method as claimed in claim 1, in which the non-human mammal is a rabbit.

8. A method as claimed in any one of claims 1 to 7, in which the mariner-like element is the transposable element mariner from Drosophila mauritiana.

9. A method as claimed in any one of claims 1 to 8, in which the mariner-like element containing the transgene is introduced into the cell in a construct.

10. A method as claimed in any one of the preceding claims, in which the introduction of the mariner-like element (MLE) is achieved by injection of the MLE
into the cytoplasm or into the pronucleus of a zygote or the nucleus of an avian or non-human mammalian embryo cell, or in which introduction of the MLE is achieved by electroporation or using liposomes.

11. A method for the preparation of a transgenic avian or non-human mammalian embryo comprising the step of introducing a mariner-like element (MLE) containing a transgene into an adult cell, optionally including co-introduction of exogenous transposase protein or DNA encoding a transposase.

12. A method for the preparation of a transgenic avian or non-human mammalian embryo comprising the step of introducing a mariner-like element (MLE) containing a transgene into a foetal cell, optionally including co-introduction of exogenous transposase protein or DNA encoding a transposase.

13. A method as claimed in any preceding claim, in which the resulting avian or non-human mammalian embryo is prepared by (i) removing the nucleus of the cell following introduction of the mariner-like element containing the transgene and its insertion into the chromosomes, and (ii) subsequently introducing the nucleus into an enucleated oocyte which is allowed to develop.

14. A method for the preparation of an avian or non-human mammal, the method comprising the steps of:
(a) preparing an embryo according to any one of the preceding claims;
(b) causing an avian or non-human mammal to develop to term from the embryo; and, optionally (c) breeding from the avian or non-human mammal so formed in step (b)

15. A method as claimed in claim 14, in which the embryo is further manipulated prior to full development of the embryo.

16. A transgenic avian or non-human mammal prepared by a method according to claim 14 or claim 15.

17. A method for the preparation of a transgenic avian or mammalian cell comprising the step of introducing a mariner-like element (MLE) containing a transgene into an avian or mammalian cell, optionally including co-introduction of exogenous transposase protein, or a DNA or RNA sequence enclosing a transposase.

18. A mariner-like element for use in the therapy of a disease condition caused by the absence of a gene or the mutation of a gene.

19. The use of a mariner-like 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.

20. 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 avian or mammalian cell, optionally including introduction or transposase protein or DNA encoding transposase.