WO1995020653A1

WO1995020653A1 - Use of the vargula luciferase gene for transgenic embryo selection

Info

Publication number: WO1995020653A1
Application number: PCT/FR1995/000099
Authority: WO
Inventors: Jean Paul Renard; Eric Malcolm Thompson
Original assignee: Institut National De La Recherche Agronomique (Inra)
Priority date: 1994-01-31
Filing date: 1995-01-30
Publication date: 1995-08-03
Also published as: AU1581995A; FR2715665A1; ZA95659B; EP0742819A1; CA2181264A1; FR2715665B1

Abstract

Use of a gene coding for Vargula luciferase, controlled by a suitable promoter, as a reporter gene for transgenic embryon detection. Advantageously, said Vargula luciferase gene is flanked by SAR sequences.

Description

USE OF THE VARGULA LUCIFERASE GENE FOR THE SELECTION OF TRANSGENIC EMBRYOS.

The present invention relates to a process for selecting transgenic embryos. Transgenesis is widely used in mice, but still little developed in other animals, such as farm animals. This situation is explained by the high cost of this technology linked mainly to the low rate of integration of a transgene into the transcriptionally active nucleus of the egg: it is estimated that in most domestic species, less than 1% of embryos injected which develop over time after transfer to a recipient female are transgenic, whereas this rate is approximately 10% in mice. This situation implies in these species to resort to an even higher number of embryos and recipient females. As a result, the cost of the first transgenic calf produced was estimated at around 3 million francs [SEIDEL, J. Anim. Sci., 71, 26-33, (1993)].

To reduce this cost, it has been proposed to increase the rate of integration of exogenous DNA by looking for new vectors that integrate more easily into the genome of the embryo. However, this approach has not yet resulted in industrially applicable results.

Another way, more direct and potentially more interesting in the short term, would be to sort the transgenic embryos before their transfer. Indeed, in several species, it is not so much the cost of producing these embryos that constitutes the main economic obstacle, as that linked to access to a large number of recipient females once the injection has been carried out: for example, in cattle, the equivalent of approximately 150 recipients is required to produce a transgenic founder animal [HILL et al., Theriogenology, 37, 222, (1992)]. Under these conditions, the technical cost of producing microinjected blastocysts represents only only 10% of the cost of maintaining the recipient herd. Sorting the embryos before their transfer can remedy this situation and an abundant literature is devoted to this subject. All the methods proposed to date use biopsy of morula or blastocysts, associated with direct detection of the transgene after amplification by PCR. They involve manipulating the embryos individually several days after having microinjected them and in addition only allow a negative sorting by eliminating the embryos for which no signal is obtained after amplification: these represent, according to the authors, 50% [ HORVAT et al., Transgenic research, 2, 134-140 (1993)] to 80% [SEIDEL, (1993), cited above] of bovine blastocysts derived from microinjected eggs.

The use of a biopsy which only allows a diagnosis to be made on part of the manipulated morula and blastocysts comes up against two major obstacles:

- on the one hand, it compromises the possibilities of development after freezing, which constitutes a significant obstacle to the dissemination of embryos;

- on the other hand, it leads to the detection of "false positive" embryos (because of technical problems during the carrying out of the amplification) which can represent up to 10% of the embryos analyzed [HORVAT et al., ( 1993), publication cited above], as well as that of "false-transgenic" embryos [SEIDEL, (1993), publication cited above] because most methods do not allow a true distinction to be made between integrated DNA and that which does not is not and which will be gradually degraded in the later stages of embryogenesis. In cattle, around 20% of microinjected embryos at the "cell" stage then divide normally in culture, and half of the blastocysts obtained (ie 10% of injected embryos) are detected as positive [ROSCHLAU et al., J. Reprod . Fert., Suppl 58, 53-60 (1989); HORVAT et al., (1993), cited above]. However, 20% of blastocysts (and morula) from injected eggs are able to give a calf. From these data it appears that 2% of the embryos injected are likely to give birth to a transgenic animal.

It seems that this percentage is actually lower, of the order of 0.2% [HILL et al., (1992), publication cited above] to only 0.1% [SEIDEL, (1993), publication cited above]. This difference probably reflects the fact that for part of the blastocysts obtained, the DNA injected is in non-integrated form. Achieving a positive sort to identify embryos where the injected DNA has effectively integrated becomes an objective all the more interesting in practice.

The present invention proposes to remove these two obstacles, by allowing the analysis of embryos at the blastocyst (or morula) stage by a non-invasive method (which does not require micromanipulation), which is rapid because it allows batch analysis. blastocysts from microinjected eggs, retaining only those which are actually transgenic, at a stage compatible with their preservation by freezing. To this end, the inventors had the idea of carrying out the injection of a transgene simultaneously with another reporter transgene which would serve to identify the embryos where integration has actually occurred.

This however requires having a reporter gene expressing itself in the embryo in an easily detectable manner, and so that neither the expression of said gene, nor the detection of its activity compromise the survival of the embryos.

Numerous reporter genes are already used in genetic engineering to select transformed cells. Among those which are used in eukaryotic cells, mention may be made, for example, of alcohol dehydrogenase [NIELSEN and PEDERSEN, J. Exp. Zool., 257, 128-133, (1991)], the CAT enzyme, β-galactosidase or firefly luciferase. In order for these reporter systems to be compatible with an in situ analysis of living cells, they must be pretreated, either to permeabilize their membranes or to limit the background noise, which increases rapidly during the measurement in the presence of the substrates. This pretreatment involves manipulations incompatible with rapid and routine analysis. The use of reporter genes whose product is secreted in the environment could remove these obstacles. This is the case with alkaline phosphatase from the human placenta, the activity of which can be detected in the culture medium by bioluminescence. Although sensitive, this method has several major drawbacks which make it difficult to apply for the selection of transgenic embryos: it involves cultivating the embryos separately, carrying out repeated samples from the culture medium; on the other hand, the detection of the signal is obtained in two stages, which results in a rapid dilution of the signal in the surrounding medium, making in practice the implementation difficult.

So far, the only gene used as a reporter gene for sorting transgenic animals is a tyrosinase minigene which transforms albino mice into pigmented mice [OVERBEEK et al., Transgenic Research, 1, 31-37, (1991)], which has been used in mice to visualize transgenic newborns. However, this gene cannot be used for early visualization before transplanting the injected embryos.

The inventors have now selected a reporter gene, the product of which is secreted in the medium and easily detectable: it is the Vargula luciferase gene (Vargula hilgendorfii) [THOMPSON et al., Proc. Natl. Acad. Sci. USA, 86, 6567-6571, (1989); THOMPSON et al., Gene, 199-204 (1990)].

The activity of this luciferase is detectable qualitatively and quantitatively by measuring the luminescence induced in the medium, in the presence of its substrate (luciferin), and of O ₂ _

The turnover of Vargula luciferase is much higher than that of other known luciferases (1600 molecules per minute whereas it is only a few tens for fly luciferase), which increases the sensitivity accordingly detection, and is an important advantage in luminescence imaging. The inventors have further found that the expression of the Vargula luciferase gene was detectable in the embryo from the earliest stages of development (stage 2 cells), and did not interfere with subsequent development. The inventors have further noted that the detection of the light signal resulting from the activity of Vargula luciferase could be used to separate at the blastocyst stage the embryos which express the transgene in the non-integrated state (transient expression) from those where integration has effectively taken place (transgenic embryo).

The subject of the present invention is the use of a gene coding for Vargula luciferase, placed under the control of an appropriate promoter, as a reporter gene for the detection of transgenic embryos. An "appropriate promoter" is a functional promoter in eukaryotic cells, in particular mammalian cells; any type of promoter which can be used for obtaining transgenic animals may be suitable; by way of example, mention may be made of the cytomegalovirus promoter, the metallothionein promoter, that of thymidine kinase, that of the elongation factor EF1.

For an optimal implementation of the object of the invention, a strong promoter will preferably be chosen, in order to obtain a significant expression of the luciferase, which increases the sensitivity of the method.

The inventors have obtained several lines of homozygous transgenic mice in which the coding sequences of the Vargula luciferase gene are placed under the dependence of the promoter HSP 70-1 [HUNT and CALDER OOD, Gene, 87, 199-204, (1990) ]. This construction will be referred to below as HSP-70-1-VL.

The luciferase activity in the culture medium of blastocysts taken from transgenic mice having only 2 copies of the HSP-70-1-VL construct can be directly viewed, after simple addition of the luciferase substrate, under an optical microscope equipped with 'a camera.

This visualization is rapid (10 to 30 seconds of image accumulation is sufficient), can be carried out at several stages of evolution of the blastocyst and is compatible with the simultaneous screening of several embryos.

The inventors have also found that when the promoter / gene of Vargula luciferase is flanked in 5 'and 3' of SAR sequences, the average level of luciferase activity measured in the culture media of blastocysts carrying this construction integrated into the genome is increased significantly (about 10 times).

SAR sequences (Scaffold Attached Regions) also known as MAR sequences (Matrix Attached Regions) have been demonstrated 5 'and 3' from the gene for human β interferon, [BODE and MAASS, Biochemistry, 27, 4706-4711 ( 1988)]. Other SAR sequences have also been demonstrated in 3 ′ and 5 ′ of various genes: [KAS AND CHASIN, J. Mol. Biol. 198: 677-692, (1987); GASSER AND LAEMMLI, Cell 46: 521-530, (1986); COCKERILL AND GARRARD, Cell 44: 273-282, (1986); PHI-VAN AND STRATLING, EMBO, 7: 655-664, (1988); JARMAN AND HIGGS, EMBO 7: 3337-3344 (1988)].

It has been shown that these SAR sequences increase, regardless of their orientation, the transcription of the genes which they flank, when these are in the integrated state, but on the other hand repress the expression of these genes when they are carried by a DNA molecule in linearized form [KLEHR et al., Biochemistry, 30, 1264-1270 (1991)].

On the other hand, the inventors have found that these same SAR sequences do not exert a stimulating effect on the expression of the gene which they flank when the DNA carrying this construct is present in the non-integrated state, but in supercoiled form, while the observations made by KLEHR et al concerned only the unrolled linear DNA.

The presence of SAR sequences therefore makes it possible to increase the difference between the level of luciferase activity due to the transient expression, and that due to the expression of the integrated DNA.

The inventors have furthermore found that the activity of stimulating the expression of the transgene by the flanking SAR sequences, which is observed in the embryo, no longer exerts in the differentiated tissues of the newborn and transgenic animals. adults, which has the advantage of limiting the risks of interference of a high production of luciferase with the physiology of animals.

The heterologous gene whose integration is to be detected can be placed on the same DNA fragment as the chimeric reporter gene according to the invention. It can also be located on another DNA fragment which is then co-injected with the fragment carrying the chimeric reporter gene. It has in fact been observed that coinjection into the egg of mice of different DNAs is compatible with the normal expression of each DNA in the adult animal [EINAT et al. Genes Devel., 1, 1075-1084, (1987), OVERBECK et al., Transgenic Research, 1, 31-37, (1991)]. The heterologous gene can optionally be placed under the control of the same promoter as the luciferase gene; however, it is preferable that it be provided with its own control sequences (promoter, enhancer, etc.). The present invention relates to a chimeric gene, usable as a reporter gene to detect the insertion of a heterologous gene, characterized in that it comprises:

a sequence coding for Vargula luciferase, which sequence is placed under the control of an appropriate promoter;

- the whole being flanked in 5 ′ and 3 ′ of SAR sequences.

The inventors established, with different constructions in accordance with the invention and comprising SAR sequences, several lines of transgenic mice. By quantifying the luciferase activity of their embryos, it is possible to finely measure the effect of disturbances of the chromatin environment on the activity of the embryonic nucleus at different stages of the preimplantation period. The present invention can be implemented to make it usable routinely in different animal species, including mammals, but also other animals such as fish, and in particular in farm animals, sorting, at different stages. of development, effectively transgenic embryos after microinjection at the pronauau stage.

In mice, it is known that approximately half of the blastocysts positive for the presence of exogenous DNA after injection at the pronal stage (and detection after amplification by PCR) are effectively transgenic. This species can be used to calibrate the constructs by defining, during the observation of the embryos under luminescence microscopy, a threshold time for recording the images obtained, time from which only the activity of the transgenic morulas or blastocysts will be effectively taken. into account. The inventors have also obtained several lines of pluripotent and totipotent cells, derived from transgenic animals according to the invention.

The present invention will be better understood with the aid of the additional description which follows, which refers to examples of construction of transgenic animals in accordance with the invention, and of detection of luciferase activity in embryos of these animals .

It should be understood, however, that these examples are given solely by way of illustration of the subject of the invention of which they do not in any way constitute a limitation.

EXAMPLE 1 CONSTRUCTION OF A PLASMID COMPRISING THE VARGULA LUCIFERASE GENE UNDER CONTROL OF THE HSP70-1 PROMOTER

A 800 bp HincII fragment comprising the essential elements of the promoter of the mouse HSP70-1 gene [HUNT and CALDERWOOD, Gene, 87, 199-204, (1990)], namely 4 elements inducible by thermal shock, arranged in tandem , and allowing the binding of specific transcription factors of thermal shock; a CCAAT box in reverse orientation; a TATA box in reverse orientation; two binding sites for the transcription factor SP1, and one binding site for the transcription factor AP2, was obtained from a BglII-NcoI fragment isolated from an EcoRI fragment of the plasmid pM2.3 from HUNT and CALDERWOOD (Gene 87, 199-204, 1990) and cloned at the HincII site of the plasmid pBluescript SK (STRATAGENE). The plasmid thus obtained was designated pBHS

A 2000 bp HindIII-BamHI fragment coding for Vargula luciferase was obtained from the plasmid PRSWL [THOMSON et al., Proc. Natl. Acad. Sci. USA, 86, 6567-6571 (1989)] and cloned between the Hind III and BamHI sites of the vector pBHS. The plasmid obtained in this way was designated pBHSVL. EXAMPLE 2 - CONSTRUCTION OF A PLASMID COMPRISING 3 'and 5' SAR SEQUENCES

On the other hand, 5 'and 3' SAR sequences were obtained as follows:

From a human cDNA library, it is possible to amplify the sequence of the gene coding for the β interferon, using appropriate primers, derived from the sequence published by OHNO and TANIGUCHI,

[Proc. Natl. Acad. Sci. 78, 5305-5309, (1981)]. After purification of the amplified fragment by electrophoresis on agarose gel, 32 P-labeled probes are obtained by random priming from this fragment; these probes are then used to screen a DNA library human genomics, in order to obtain genomic clones comprising the 5 'and 3' flanking sequences of the interferonβ gene.

From the genomic clones obtained, and based on the restriction map of the SAR regions 3 'and 5' of the interferon β gene which has been published by BODE and MAASS, [Biochemistry, 27: 4706-4711, (1988)], a 2.2 kb EcoRI fragment which comprises the 5 ′ SAR sequence is subcloned and a 3 kb BglIII fragment which comprises the 3 ′ SAR sequence.

The 3 kb BglIII fragment was cloned into the BamHI site of the plasmid pBHSVL described above to give the plasmid pSHSVL. On the other hand, the 2.2 kb EcoRI fragment previously provided at each of its ends with XhoI-EcoRI adapters was inserted into the Xhol site of pSHSVL, 5 ′ of the HSP70.1 promoter.

The plasmid obtained was designated pSHSVLS. EXAMPLE 3 - OBTAINING TRANSGENIC MICE

The transgenic mice were obtained from the two different constructs, pBHSVL (SAR-) and pSHSVLS (SAR ⁺ ) obtained according to the protocols described in Examples 1 and 2 respectively.

The Kpnl-BamHI fragments of pBHSVL and Kpnl-Notl of pSHSVLS were purified from agarose gels (GENE CLEAN kit; BIO 101), and the DNA was injected at a concentration of 2ng / μl into the pronuclei. fertilized eggs obtained by crossing F1 hybrids (male and female) C57BL6 / CDA.

The transgenic founders were identified by PCR using primers specific for the Vargula luciferase gene. The number of copies of the gene was determined by densitometric analysis of quantitatively performed Southern blots. After identifying the founders, they were crossed with each other in order to obtain animals homozygous for the transgene. EXAMPLE 4 - OBTAINING EMBRYOS AND TISSUES OF TRANSGENIC ANIMALS

1) Embryos

Over-ovulation was induced in female FI hybrids C57BL6 / CBA, by injecting 10 U of pregnant mare serum gonadotropin (PMSG; INTERVET) in 0.1 ml of physiological saline, this injection being followed 46 to 48 hours later by l injection of 5U of human chorionic gonadotropin (HCG; INTERVET), in 0.1 ml of the same solution. Treated females were mated with transgenic males homozygous for the transgene, and 1-cell stage embryos were recovered from the oviducts 21 to 23 hours after the injection of HCG. The embryos were placed in drops of M16 medium [HOGAN et al., Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring harbor, N.Y. (1986)] at a density of approximately 1 embryo / ml of culture medium. The embryos were cultured in an incubator, at a temperature of 37 "C and in the presence of 8% C02.

The constitutive expression is measured throughout the culture period by taking a little medium at desired time intervals, and replacing it each time with fresh medium. Each fraction removed is immediately frozen at -80 "C until detection of luciferase activity.

To determine the expression inducible at the blastocyst stage, the culture dishes containing blastocysts (101-112 hours after HCG) sealed with a sealing film, were immersed in a water bath at 43 ^° C for 30 minutes. The sealing film has been removed, and the dishes placed again in the incubator at 37 "C. The Vargula luciferase begins to accumulate in the medium after approximately 3 hours. The kinetics of accumulation can be followed by of successive samples, or measure the total amount accumulated at the end of the culture.

2) Tissues of transgenic animals Females of generation F1 of the hybrid line C57BL6 / CBA were mated with males homozygous for the transgene. The gestations were completed, and two batches of animals were chosen for the experiments: either mice of 1 day, or adults of 5 weeks. The animals were killed and tissue samples were taken.

EXAMPLE 5 DETERMINATION OF LUCIFERASE ACTIVITY

The activity of the luciferase secreted by the embryos is analyzed by two different methods: 1st method: An aliquot of the culture medium of the embryos is taken. This aliquot is then diluted with 200 mM Tris-HCl, pH 7.6, to a volume of 100 μl. The sample thus diluted is placed at the bottom of a 5 ml measuring tube (SARSTEDT) and introduced into the measuring chamber of a LUMAT LB 9501 photometer (BERTHOLD Instruments). Luciferin, substrate of Vargula luciferase, is prepared according to one of the protocols described by TSUJI [Methods Enzy ol. 57, 364-372, (1978)], or by KISHI et al., [Tetrahedron Lett., 3445-3450, (1966)], INOUE et al. [Tetrahedron Lett., 1609-1610, (1969)], SUGIURA et al., [Yakugaku Zasshi, 90, 707-710 (1970]. A 100 nM solution of luciferase in 200 mM sodium phosphate buffer at pH 6, 8 is placed in a glass container in which argon is bubbled continuously (in order to prevent auto-oxidation of the luciferin). To measure the luciferase activity of the sample, 100 μl of the solution of Luciferin is injected into the tube comprising the sample, and the intensity of the light emitted is measured by the photometer. 2nd method: The luciferase activity can also be viewed directly under a microscope, equipped with a camera (Argus-50, HAMMAMATSU instruments). 2 μl drops of a 50 μM solution of luciferin in PBS buffer are placed in the microscopy chamber, and covered with oil. The embryos are introduced into these drops, and the whole is placed under an inversion microscope equipped with a camera. After exposure for a period of 10 to 30 seconds, the luminescence emitted by the transgenic embryos which have integrated the luciferase gene is detected, which allows the selection of the embryos. RESULTS: It is known that the HSP70-1 gene expresses constitutively in the embryo at the 2-cell stage and is inducible by heat shock at the blastocyte stage [BENSAUDE et al., Nature, 305, 331-333, (1983)].

The in vitro development of transgenic embryos containing the constructs with or without the SAR sequences is the same as that of the non-transgenic embryos used as control. The SAR + lines show a higher expression than the SAR- lines both at the 2-cell stage and at the blastocyte stage. At the 2-cell stage, all the SAR- lines show practically the same basic level of expression, which does not appear to depend on the number of copies. In contrast, transgenic SAR + lines express luciferase at higher levels, and expression is a function of the number of copies. On average, an expression 6 times higher is observed in the SAR + group than in the SAR- group. When this expression is reduced to the number of copies, there is an increase 2.5 times greater in the SAR + group than in the SAR- group. At the blastocyte stage, the same type of observations were made: the SAR- lines express the luciferase independently of the number of copies, whereas in SAR + lines the expression of luciferase is positively correlated with the number of copies. Copy expression in SAR + lines is 4.1 times greater than copy expression in SAR- lines.

Expression results in tissues from newborn animals and adults show that there is no longer stimulation of transgene expression by flanking SAR sequences in differentiated tissues, with the exception however tail biopsies, in which it appears that the SAR sequences still confer some degree of stimulation of transgene expression. In addition, the expression of the transgene decreases from the newborn mouse to the adult mouse in the case of the SAR- lines, whereas in the case of the SAR + lines it is substantially equivalent whatever the age of the animal.

Claims

1) Use of a gene coding for Vargula luciferase, placed under the control of an appropriate promoter, as a reporter gene for the detection of transgenic embryos.

2) Use according to claim 1, characterized in that said promoter is the HSP70-1 promoter.

3) Use according to any one of claims 1 or 2, characterized in that the promoter / gene of Vargula luciferase is flanked in 5 'and 3' of SAR sequences.

4) Chimeric gene, usable as a reporter gene to detect the insertion of a heterologous gene, characterized in that it comprises:

- the whole being flanked in 5 ′ and 3 ′ of SAR sequences.

5) Transgenic animal, characterized in that it comprises at least one copy of the gene coding for Vargula luciferase, under the control of an appropriate promoter. 6) Transgenic animal according to claim

5, characterized in that it comprises a chimeric gene according to claim 4.

7) Transgenic animal according to any one of claims 5 or 6 characterized in that said animal is a mammal.

8) Embryo of an animal according to any one of claims 5 to 7.

9). Cell line, characterized in that it is derived from an animal according to any one of claims 5 to 7, and in that each cell of said line comprises at least one copy of the coding gene for Vargula luciferase, under the control of an appropriate promoter.