CA2264450A1

CA2264450A1 - Methods for monitoring heterologous sex chromosomes

Info

Publication number: CA2264450A1
Application number: CA 2264450
Authority: CA
Inventors: Andras Nagy; Anna-Katerina Hadjantonakis
Original assignee: Individual
Current assignee: Mount Sinai Hospital Corp
Priority date: 1998-03-05
Filing date: 1999-03-05
Publication date: 1999-09-05

Abstract

Pluripotent cells comprising a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into a heterologous sex chromosome in the cell; embryos and transgenic animals produced using the pluripotent cells; and, the uses of such cells, embryos, and animals are described.

Description

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MSH File No. GFP CA
TITLE: Methods for Monitoring Heterologous Sex Chromosomes FIELD OF THE INVENTION
The invention relates to methods for monitoring heterologous sex chromosomes.
The invention also relates to pluripotent cells comprising a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into a heterologous sex chromosome in the cell; embryos and transgenic animals produced using the pluripotent cells;
and, the uses of such cells, embryos, and animals, in particular in monitoring heterologous sex chromosomes.
BACKGROUND OF THE INVENTION
In many species the male and female sex is determined by the segregation of specialized chromosomes i.e. sex chromosomes. There has been a long time interest to control this random process in many species of wild, farm, and laboratory animals. The reasons for controlling sexing include reducing the incidence of sex-linked genetic disorders, and more efficient animal production. For example, the bovine dairy and beef agriculture sectors need female and male animals respectively, and the undesired animal is culled.
Selection of animals of a desired sex has been carried out by sexing of preimplantation embryos, or separation of X and Y bearing spermatozoa.
Preimplantation embryo sex selection has been accomplished using karyotyping, amplification of Y
chromosome specific nucleotide sequences, or immunological methods. The first two methods are invasive methods since they involve embryo micromanipulation.
Immunological methods are typically based on immunodetection of a male-specific marker. Eichwald and Silmser (1955, Transplant Bull 2:148) found that within the inbred mouse strain C57BL/6, skin transplants from males to females were rejected, whereas transplants from males to males, females to males and females to females within the same strain were tolerated. These results were attributed to an antigen coded for by a Y-linked gene, and the system came to be known as H-Y (histocompatibility locus on the Y
chromosome) (Hauscha (1955, Transplant Bull 2:154). Antibodies specific for H-Y are used in sexing studies e.g. enzyme-linked immunoabsorbent assay (ELISA) methods [Bradley et al. (1987, Hum. Genet. 76:352) and Brunner and Wachtel (1988, J. Immunol.
Methods 106:49). See also Epstein, (1980, Tiss. Antigens 15:63); Anderson, (1987, Theriogenology 27:81); Wachtel, (1988, Fert. Ster. 50:355); Avery and Schmidt, (1989, Acta.
Vet. Scand.
30:155) for the use of H-Y antibodies to sex embryos]. However, it is uncertain that H-Y is preferentially expressed on Y-bearing sperm (e.g. Hendricksen et al. 1993, Mol. Reprod.
Devel. 35:189) and, some investigators have concluded that differences between the two classes of sperm can not be detected immunologically (Windsor et al. (1993, Reprod. Fert.
Dev. 5:155).
Further, a "female protein" which is hormone-dependent and therefore unlikely to be found in blastocysts or sperm has been reported by Brown et al. (1991, Nature 349: 38).
An XX-specific molecule which is an mRNA molecule transcribed by the "inactivated" X, and therefore only produced in females in somatic tissues has also been reported by Coe (1977, Proc. Nat. Acad. Sci. 74: 730).
Cell markers have been used in transgenic animal production to select embryos that have an integrated transgene. Typical selection methods employ reporter genes such as luciferase, ~3-galactosidase, and alkaline phosphatase that are not particularly useful since they require harmful substrates for their detection. Other methods involve PCR
analysis of blastomere biopsies which requires time and complicated micromanipulation.
There is also a high risk that an embryo will be damaged using these selection methods.
Non-invasive methods have been reported for selection of transgene-integrated embryos using green fluorescent protein (GFP) as a marker (Takada, T., et al, Nature Biotechnology 15:458, 1997). The method involves microinjecting embryos with a GFP
transgene, identifying GFP-positive blastocysts, and transferring the blastocysts to uteri of pseudopregnant females to produce transgenic mice. The method has a number of disadvantages. In some studies, it has not been possible to obtain fetuses from blastocysts that strongly expressed GFP (Takada, T. et al, supra). Fetus production rates have been reported to be less than 25% and have been postulated to be the result of the damage caused by microinjection and gene integration. Further, in a number of studies microinjection of wild-type GFP has not resulted in ubiquitous expression of GFP (H. Niwa et al Gene 108:193-199, 1991; M. Ikawa, FEBS Lett 375:125-128, 1995; and M. Ikawa, et al, Dev.
Growth Diff 37:455-459, 1995). Others have used mutant GFPs and have reported a visible signal in all tissues with the exception of erythrocytes and hair (M. Okabe et al, FEBS
Letters 407:313-319, 1997). One mutant form of GFP (MmGF'P) has been used to make an embryonic stem cell line expressing MmGFP, and the cells have been introduced into mouse embryos (Zernicka-Goetz M., et al Development 124:1133-1137, 1997).
Cell markers that require substrates have been used to monitor X chromosome activity at the single cell level. In particular, an X-linked lacZ transgene has been used to examine the progression of X-inactivation in different somatic lineages (Tam, P. et al Development 120:2925-2932, 1994).
SUMMARY OF THE INVENTION
Broadly stated the present invention relates to a pluripotent cell comprising a nucleic acid sequence encoding a fluroescent protein marker selectively integrated into a heterologous sex chromosome in the cell. The invention also provides a pluripotent cell line comprising pluripotent cells of the invention. In particular an embryonic stem cell line is provided comprising embryonic stem cells of the invention. Further, the invention provides a chimeric embryo comprising pluripotent cells of the invention, in particular embryonic stem cells of the invention.
The invention also provides a method of producing a pluripotent cell that has a heterologous sex chromosome linked fluorescent protein marker comprising introducing into a pluripotent cell a nucleic acid sequence encoding a fluorescent protein marker, and optionally a nucleic sequence encoding a protein of interest, under conditions so that the fluorescent protein marker is selectively integrated into a heterologous sex chromosome in the cell.
In an embodiment of the invention a method is provided for producing an embryonic stem cell that has an X-linked fluorescent protein marker comprising introducing into embryonic stem cells a nucleic acid sequence encoding a fluorescent protein marker, and optionally a nucleic sequence encoding a protein of interest, under conditions so that the fluorescent protein marker is selectively integrated into the X chromosome in the cell.
The invention also contemplates a method of producing a chimeric embryo that develops into a non-human animal that transmits to its progeny a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in the animal's cells comprising:
(a) providing pluripotent cells, in particular embryonic stem cells, comprising a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in the cell; and (b) introducing the pluripotent cells in an embryo or aggregating the pluripotent cells with an embryo to produce a chimeric embryo.
A chimeric embryo may be implanted into a pseudopregnant foster mother, and allowed to grow to term to produce a non-human transgenic animal that is capable of transmitting the nucleic acid sequence only to female or male progeny. Therefore, the invention also contemplates a non-human transgenic animal having a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in its cells.
The invention still further provides a method for producing a non-human animal having a fluorescent protein marker integrated into one of the heterologous sex chromosomes in its cells comprising;
(a) selecting an embryo that contains cells with a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in the cells;
(b) allowing the embryo to develop to term to produce a non-human animal; and (c) optionally breeding the non-human animal and selecting progeny that express the fluorescent protein marker.
In an embodiment of the invention a method is contemplated for producing a non-human animal, preferably a rodent, having a fluorescent protein marker selectively integrated into one of the sex chromosomes in its cells comprising the steps of:
(a) introducing into an embryo of the animal, pluripotent stem cells comprising a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in the cells;
(b) allowing the embryo to develop to term to produce a non-human animal having a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in its cells; and (c) optionally breeding the animal wherein one of the male or female progeny express the fluorescent protein marker.
The non-human animals of the invention may be bred to normal animals or other animals carrying a second transgene(s) to create an animal carrying two transgenes i.e. a double transgenic animal.
The invention also provides a method of monitoring expression of a protein of interest linked to one of the heterologous sex chromosomes in a non-human animal comprising:
(a) introducing into pluripotent cells a transgene comprising a DNA sequence encoding a protein of interest and a sequence encoding a fluorescent protein marker under conditions so that the fluorescent protein marker is selectively integrated into a heterologous sex chromosome in the cells;
(b) culturing the pluripotent cells in conditions permitting expression of the fluorescent marker protein and the gene of interest; wherein the pluripotent cells expressing the fluorescent marker protein express the gene of interest; and (c) monitoring the expression of the gene by monitoring expression of the fluorescent protein marker in the pluripotent cells.
The pluripotent cells (e.g. embryonic stem cells), may be injected into a blastocyst or aggregated with an early stage embryo to produce a chimeric embryo and the expression of the protein may be monitored by monitoring expression of the fluorescent protein marker in the chimera or in the growing chimera. The chimera may be implanted into a pseudopregnant foster animal and the expression of the gene may be monitored by monitoring expression of the fluorescent protein marker in the growing chimera or in the resultant transgenic animal.
The invention also provides a method of selecting embryonic stem cells containing a preselected heterologous sex chromosome comprising (a) introducing into embryonic stem cells a transgene comprising a sequence encoding a fluorescent protein marker under conditions so that the fluorescent protein marker is selectively integrated into the heterologous sex chromosomes in the cells;
(b) culturing the embryonic stem cells in conditions permitting expression of the fluorescent protein marker; and (c) selecting embryonic stem cells containing the heterologous sex chromosome by detecting embryonic stem cells expressing the fluorescent protein marker.
The invention further provides a method of selecting male or female embryos comprising: producing embryos containing a fluorescent protein marker selectively integrated into a preselected heterologous sex chromosome; and selecting male or female embryos depending on whether the embryos express the fluorescent protein marker.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure lA is a photograph showing the expression of EGFP in ES cells;
Figure 1B is a photograph showing the expression of EGFP in drug-resistant colonies, exhibiting ubiquitous EGFP expression;
Figure 1C is a photograph showing the expression of EGFP in differentiated ES
cells;
Figure 1D is a photograph showing embryoid bodies exhibiting ubiquitous transgene activity;
Figure 2A is a photograph showing ES cells colonizing part of, and the majority of morula stage embryos;
Figure 2B is a photograph showing ES cells colonizing most of the ICM but not the trophectoderm of a blastocyst stage embryo;
Figure 2C is a photograph of ES cells colonizing a proportion of the inner cell mass (ICM) of a blastocyst;
Figure 2D is a photograph of ES cells colonizing a proportion of the inner cell mass of a blastocyst;
Figure 2E is a photograph of ES cells localized to the majority of the ICM
with a single cell in the trophectoderm of a blastocyst;
Figure 2F is a photograph of ES cells localized to part of the ICM with two cells seen in the trophectoderm;
Figure 3A is a schematic diagram of a method detailing the technique of diploid green ES cell wild type embryo aggregations;
Figure 3B is a photograph showing the aggregation of a clump of green fluorescent ES cells with a wild-type embryo to form a blastocyst after overnight incubation;
Figure 3C is a photograph showing the aggregation of a clump of green fluorescent ES cells with a wild-type embryo to form a blastocyst after overnight incubation;
Figure 3D is a photograph showing the aggregation of a clump of green fluorescent ES cells with a wild-type embryo to form a blastocyst after overnight incubation;
Figure 3E is a photograph showing the aggregation of a clump of green fluorescent ES cells with a wild-type embryo to form a blastocyst after overnight incubation, with preferential contribution of the ES cells to the inner cell mass;
Figure 4A is a photograph of a postimplantation embryo showing the ventral view of a late headfold/presomite stage (E8) embryo;
Figure 4B is a photograph of a postimplantation embryo showing a lateral view of an early somite stage (E8.5) embryo;
Figure 4C is a photograph of a postimplantation embryo showing a dorsal view of an E8.5 embryo;
Figure 4D is a photograph of a postimplantation embryo showing a lateral view of the head and forelimb of a new-born chimera;
Figure 5A is a photograph of one- to four-cell stage embryos exhibiting no transgene expression;
Figure 5B is a photograph of embryos showing initiation of expression at the morula stage;
Figure 5C is a photograph of E3.5 embryos in dark-field;
Figure 5D is a photograph of E3.5 embryos in bright-field;
Figure 5E is a photograph of two-cell and morula stage green-fluorescent embryos in bright-field;
Figure 5F is a photograph of two-cell and morula stage green-fluorescent embryos in dark-field;
Figure 5G is a photograph of blastocyst stage embryos in dark-field;
Figure 5H is a photograph comparing embryos derived from a hemizygous male crossed to a wild-type female and a wild-type male crossed to a hemizygous female;
Figure 6A is a photograph of an E4.75 newly hatched and implanted blastocyst attached to decidual tissue;
Figure 6B is a photograph showing B5/EGFP expression in an E5 embryo;
Figure 6C is a photograph showing B5/EGFP expression in an E5.5 embryo;
Figure 6D is a photograph showing B5/EGFP expression in an E9 embryo with associated ectoplacental cone partially contained within the deciduum;
Figure 6E is a photograph showing B5/EGFP expression in an E9 extraembryonic _g_ membrane;
Figure 6F is a photograph of two E9.5 littermates in bright-field;
Figure 6G is a photograph in dark-field of the two embryos shown in Figure 6F;
Figure 6H is a photograph showing BSBGFP expression in an E11.5 embryo with placenta;
Figure 6I is a photograph showing B5/EGFP expression in an E11.5 embryo heart and lungs;
Figure 6J is a photograph showing B5/EGFP expression in an E11.5 embryo kidney rudiment and intestine;
Figure 7A is a photograph showing ubiquitous green fluorescence in the head and forelimb of a new-born B5/EGFP hemizygote pup among non-transgenic littermates;
Figure 7B is a photograph showing ubiquitous green fluorescence in a new-born BSBGFP hemizygote pup and non-transgenic littermate;
Figure 7C is a photograph showing ubiquitous green fluorescence in 1-week-old transgenic and non-transgenic pups;
Figure 7D is a photograph showing ubiquitous green fluorescence in an anterior view of a 3-week-old albino B5/EGFP mouse;
Figure 7E is a photograph showing ubiquitous green fluorescence in a posterior view of a 3-week-old albino B5/EGFP mouse;
Figure 7F is a photograph of a posterior view of the tails of 2-month albino, chinchilla, agouti mice illustrating pigment-dependent fluorescence;
Figure 7G is a photograph of a dorsal view of an incision made in a 3-week-old BSBGFP mouse body with skin removed to reveal fluorescence in the body;
Figure 7H is a photograph of brains from 3-week-old transgenic mouse and non-transgenic littermates;
Figure 7I is a photograph of kidneys from 3-week-old transgenic mouse and non-transgenic littermates;
Figure 7J is a photograph of livers from 3-week-old transgenic mouse and non-transgenic littermates;
Figure 7K is a photograph of pancreas from 3-week-old transgenic mouse and non-transgenic littermates;
Figure 7L is a photograph of intestines from 3-week-old transgenic mouse and non-transgenic littermates;

Figure 8A is a schematic diagram detailing the tetraploid technique;
Figure 8B is a photograph showing the progression of an aggregation between green ES cells-wild-type embryo to the blastocyst stage;
Figure 8C is a photograph showing the progression of an aggregation between green ES cells-wild-type embryo to the blastocyst stage;
Figure 8D is a photograph showing the progression of an aggregation between green ES cells-wild-type embryo to the blastocyst stage;
Figure 8E is a photograph showing the progression of an aggregation between green ES cells-wild-type embryo to the blastocyst stage;
Figure 9A is a photograph of a green fluorescent E9 embryo with non-fluorescent extra-embryonic membranes;
Figure 9B is a photograph of a green fluorescent E11 embryo with mosiac yolk sac and non-fluorescent placenta;
Figure 9C is a photograph of an E8.5 embryo with green fluorescent extraembryonic membranes;
Figure 9D is a photograph of an E11 embryo with mosaic yolk sac and fluorescent placenta;
Figure l0A is a photograph of new-born pups from a cross between a BS/EGFP
hemizygous male and a wild-type ICR female;
Figure lOB is a photograph of mice from a litter of pups derived from an intercross between a male and female both of which were hemizygous for the transgene;
Figure lOC is a photograph of a close view of pups hemizygous and homozygous for the transgene;
Figure lOD is a photograph of the tail tips from E11.5 embryos homozygous, hemizygous, and non-transgenic;
Figure 11A is a photograph showing a litter of F1 offspring derived from a cross between a D4/XEGFP transgenic male and a wild type female;
Figure 11B is a photograph of 6 pups from a litter derived from a cross between a hemizygous female and wild-type male;
Figure 12A is a photograph of an E6.5 embryo obtained from matings between a D4/XEFGP transgenic male and a wild-type ICR female, and a non-transgenic embryo;
Figure 12B is a photograph of an E8.5 embryo obtained from matings between a D4/XEFGP transgenic male and a wild-type ICR female, and a non-transgenic embryo;

Figure l2Cis a photograph of an E10.5 embryo obtained from matings between a D4/XEFGP transgenic male and a wild-type ICR female, and a non-transgenic embryo Figure 13A is a photograph of a pool of three litters of E3.5 embryos derived from natural matings between transgenic males and wild type females in bright field, where green fluorescent embryos are female and the non-fluorescent embryos are male;
Figure 13B is a photograph of a pool of three litters of E3.5 embryos derived from natural matings between transgenic males and wild type females in dark field with background illumination, where green fluorescent embryos are female and the non-fluorescent embryos are male;
Figure 13C is a photograph of a pool of three litters of E3.5 embryos derived from natural matings between transgenic males and wild type females in full dark field, where green fluorescent embryos are female and the non-fluorescent embryos are male;
Figure 13D is a photograph of color and sex pooled embryos which gave rise to single sex litters after transfer to surrogate females;
Figure 14A is a photograph in bright-field of litters of E3.5 embryos derived from a cross between a D4/XEFGP transgenic male and a wild-type ICR female;
Figure 14B is a photograph in dark field of litters of E3.5 embryos derived from a cross between a D4/XEFGP transgenic male and a wild-type ICR female;
Figure 14C is a photograph of litters of E10 embryos derived from a cross between a D4/XEFGP transgenic male and a wild-type ICR female; and Figure 14D is a gel showing PCR genotyping for the presence of a Y
chromosome using primers for Sry and a control assay for the presence of an autosomal gene using primers for the myogenin gene.
DETAILED DESCRIPTION OF THE INVENTION
As hereinbefore mentioned, the present invention relates to a pluripotent cell comprising a nucleic acid sequence encoding a fluroescent protein marker selectively integrated into one of the heterologous sex chromosomes in the cell.
The pluripotent cell may be any totoipotent, pluripotent embryonic/somatic cells, embryonic stem cells and totipotent somatic cell cultures, which are all germline compatible.
Primary isolates of embryonic stem cells may be used that are obtained directly from embryos such as the CCE cell line (Robertson, E.J. In: Current Communications in Molecular Biology, Capecchi, M.R. (ed), Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), p39-44) or from clonal isolation of embryonic stem cells from such cell lines (Schwartzberg, P.A. et al., Science 246:799-803, 1989; E.J. Robertson In:
Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (E.J.Robertson, Ed.), IRL
Press, Oxford, 1987). Clonally selected embryonic stem cells are generally more effective in producing transgenic animals since they have a greater efficiency for differentiating into an animal.
Examples of suitable embryonic stem cells are the R1 cell line (Nagy A.. et al, Proc Natl Acad Sci 90:8424-8428, 1993 and Wood et al, Nature 365:87-89, 1993).
Pluripotent cells may be obtained from any vertebrate species (i.e. mammals, birds, reptiles, amphibians, and fishes); however, cells derived or isolated from mammals such as rodents (e.g. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish, pigs, cattle, primates, and humans are preferred.
The fluorescent protein marker may be a Green Fluorescent Protein (GFP) from the jellyfish A. victoria, or a variant thereof that retains its fluorescent properties when expressed in vertebrate cells. Variants of GFP may be selected that have longer wavelengths of excitation and emission, are more ubiquitously expressed, have increased thermostability, and/or exhibit stronger fluorescent signals compared to wild-type GFP.
Examples of GFP
variants include a variant of GFP having a Ser65Thr mutation of GFP (S65T) that has longer wavelengths of excitation and emission, 490nm and 510nm, respectively, compared to wild-type GFP (400nm and 475nm); a blue fluorescent variant of GFP (e.g. Y66H-GFP) (Heim et al, Proc. Natl. Acad. Sci. 91:12501, 1994), MmGFP (M. Zernicka-Goetz et al, Development 124:1133-1137, 1997) enhanced GFP ("EGFP") (Okabe, M. et al, FEBS
Letters 407:313-319, 1997; Clontech, Cal.), or a red shifted variant, EYFP
(yellow fluorescent protein; excitation max. 513 nm). Other GFP variants are described on the worldwide web at the Structural Classification of Proteins site (http://www.pdb.bnl.gov/scop/date/ scop1.004.07.001.001.000 html). In an embodiment of the invention, the fluorescent protein marker is EGFP which has a Phe to Leu mutation at position 64 resulting in the increased stability of the protein at 37°C
and a Ser to Thr mutation at position 65 resulting in an increased fluorescence. EGFP
commercially available from Clontech incorporates a humanized codon usage rendering it "less foreign"
to mammalian transcriptional machinery and ensuring maximal gene expression. The coding sequence of Clontech's EGFP contains over 190 silent mutations that create a humanized open reading frame. Additionally sequences upstream of the EGFP have been converted to a Kozak consensus ribosome binding site, allowing for more efficient translation of the mRNA in mammalian cells.
The pluripotent cells of the invention are produced by introducing into pluripotent cells a nucleic acid sequence encoding a fluorescent protein marker and optionally a nucleic acid sequence encoding a protein of interest, under conditions so that the fluorescent protein marker is selectively integrated into a heterologous sex chromosome in the cell.
The nucleic acid sequence encoding a fluorescent protein marker may be introduced into pluripotent cells using an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.
The invention therefore contemplates an expression vector containing a nucleic acid sequence encoding a fluorescent protein marker, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence. Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art. In particular, the vector may contain a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, and an enhancer. Suitable promoters include the (3-actin promoter, elongation factor la, cytomegalovirus promoter, and lac promoter. Suitable enhancers include the cytomegalovirus enhancer element. Vectors may encode more than one fluorescent protein marker, or they may encode other reporter proteins. A
separate vector may be used to introduce another fluorescent protein marker or other reporter proteins. The vectors may be obtained commercially or assembled from the sequences described by conventional methods in the art. In an embodiment of the invention, the commercially available vectors p EGFP-1, pEGFP-N1, and pEGFP-C1 are utilized.
A nucleic acid sequence encoding a GFP or variant thereof may be introduced into embryonic stem cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
A nucleic acid sequence encoding the fluorescent protein marker (e.g. GFP or a variant thereof) is introduced into pluripotent cells under conditions so that the fluorescent protein marker is selectively integrated into a heterologous sex chromosome in the cell. The pluripotent cells are cultured using conventional methods.
The pluripotent cells may be analyzed to corroborate the introduction of a fluorescent marker protein such as GFP or a variant thereof. For example, restriction mapping, Southern hybridization assays, and the like may be performed. The polymerase chain reaction is also useful for this purpose. Appropriate primers may be chosen for the PCR reaction to screen cells for introduction of the vector having a sequence encoding a fluorescent protein marker. The primers are chosen to be complimentary to sequences to the fluorescent protein marker e.g. GFP or variants thereof.
The pluripotent cells of the invention may be used to produce a pluripotent stem cell line using conventional methods. For example, the embryonic stem cells may be cocultured with feeder cells (usually irradiated fibroblasts or stomal cells e.g. STO
cells), or cultured in medium conditioned by established teratocarcinoma stem cell lines and other cells (See E.J. Robertson In: Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (E.J.Robertson, Ed.), IRL Press, Oxford, 1987). The embryonic stem cells can be propagated without a feeder cell layer in the presence of differentiation inhibiting activity (DIA) or in the presence of factors such as leukocyte inhibitory factor (Gough, N.M. et al, Reprod. Fertil.
Dev. 1:281-288, 1989). The factors may be added to the culture or they may be produced by transforming the stromal cells so that they secrete the factor. The pluripotent cells may also be cultured in vitro to produce embryoid bodies.
The pluripotent cells of the invention may also be introduced into a blastocyst or aggregated with morula stage embryos to produce chimeric embryos. The chimeric embryos may be transferred into pseudopregnant recipients, and developed to term to yield transgenic animals. A transgenic animal of the invention may be used to breed additional animals carrying a heterologous sex-linked fluorescent marker protein. The transgenic animals of the invention may also be bred to homozygosity and/or bred with other transgenic animals. An animal exhibiting fluorescence and having germline transmission of the marker gene may be used to establish a transgenic line. A transgenic animal may be mated with a normal animal, and fertilized embryos collected and cultured.
The invention also provides a method of monitoring expression of a protein of interest linked to a heterologous sex-chromosome in a non-human animal. The method involves introducing into pluripotent cells a transgene comprising a DNA
sequence encoding a protein of interest and a sequence encoding a fluorescent protein marker under conditions so that the fluorescent protein marker is selectively integrated into the heteorlogous sex chromosomes in the cells. The DNA sequences include structural genes (e.g. DNA
sequences encoding proteins including introns and exons in the case of eukaryotes), regulatory sequences such as enhancer sequences, promoters, and the like and other regions within a genome of interest. The pluripotent cells are cultured in conditions permitting expression of the fluorescent marker protein and protein of interest. The expression of the protein is monitored by monitoring the expression of the fluorescent protein marker in the pluripotent cells.
The pluripotent cells may be aggregated with an early stage embryo or injected into a blastocyst as described herein, and the expression of the protein may be monitored by monitoring expression of the fluorescent protein marker in the chimeric embryo or in the growing chimeric embryo. The chimeric embryo may be implanted into a pseudopregnant foster animal as described herein and the expression of the gene may be monitored by monitoring expression of the fluorescent protein marker in the growing chimeric embryo or in the resultant transgenic animal.
It will be appreciated that a cell, embryo, or transgenic animal of the invention may be labeled with more than one fluorescent protein marker, or additionally labeled with one or more other reporter proteins.
The invention also provides a method of selecting pluripotent cells and embryos containing cells having a preselected heterologous sex chromosome. Pluripotent cells containing a heterologous sex-linked fluorescent marker protein may be prepared using the methods described herein and they may be cultured in conditions permitting expression of the fluorescent protein marker. Pluripotent cells containing a heterologous sex chromosome are selected by selecting embryonic stem cells expressing the fluorescent protein marker.
Embryos comprising a preselected heterologous sex chromosome produced using the methods described herein, can be selected by selecting embryos expressing the fluorescent protein marker which is linked to the heterologous sex chromosome. For example, if the fluorescent protein marker is GFP, embryos containing the heterologous sex chromosome can be readily selected using a fluorescence microscope. Selection can be observed at the morula stage or blastocyst stage. Embryos can be monitored at 470nm to 490nm during in vitro culture.
The methods of selecting embryos containing a preselected heterologous sex chromosome in their cells may be used to sex animals. The methods have application in breeding rodents for laboratory use, and for breeding farm animals such as bovines, sheep, pigs, goats etc. The methods described herein may be particularly useful in reducing sex-linked genetic disorders in farm animals. The methods may also be used to increase agricultural production. For example, the bovine dairy and beef sectors require female and male animals respectively, and the methods of the present invention enable selection of female animals.
The following non-limiting example is illustrative of the present invention:
Example 1 Materials and Methods Vectors used: pCX-EGFP, the EGFP expressing vector has been previously described (10).
It contains EGFP inserted into pCAGGS, a plasmid vector containing a chicken beta-actin promoter and CMV enhancer, beta-actin intron and rabbit ~3-globin polyadenylation signal " pPGKPuro, the puromycin resistance gene containing vectorl~ was used for co-electroporation since it carries an antibiotic resistance gene.
Creating green ES cells: Cells were co-electroporated under standard conditions with 40 80pg of linearised pCX-EGFP (BamHI or ScaI used interchangeably for linearisation) and lOmg of circular pPGKPuro. The plasmid that is to integrate into the genome is linearised, but the drug selection containing plasmid is used in a circular form since this is unlikely to integrate thereby conferring transient drug resistance. Each individual electroporation was seeded onto a single lOmm gelatin coated tissue culture plate and resulted in approximately 80-140 puromycin resistant colonies, 8-10 of which were green. Control electroporations were carried out using one or other of the plasmids singly with subsequent selection in puromycin. Selection in puromycin was initiated 48 hours after electroporation, and continued for 3-4 days thereafter. Colonies were viewed under a dissecting microscope (Leitz MZ8) equipped with a GFP illumination source (produced by Leica) and scored for green by etching the underside of the tissue culture plate with a needle.
Colonies assigned as being "green" were picked into 96 well plates for expansion and freezing.
Production of transgenic mice: Both diploid and tetraploid aggregations were performed to produce embryos using conventional techniques.

Observation of green fluorescence: The EGFP fluorescence was examined under dissecting microscopes (Leitz MZ8 and MZ12) equipped with GFP excitation sources (Leica). The excitation maxima of EGFP is at 488nm, and the emission maxima is at 507nm, the optics provided by Leica were specifically designed to detect this chromophore. Both tissue culture dishes and mouse embryos and adults could be viewed under the same optics.
Results.
Expression of EGFP in ES cells in culture: The pCX-EGFP plasmid (Okabe et al., 1997) was introduced into R1 ES cells via a co-electroporation with a drug selection encoding plasmid. Both neomycin (ploxPneo) and puromycin (pPGKPuro) encoding plasmids were used in initial trial co-electroporation studies, but eventually solely pPGKPuro was used.
The reasons for this being twofold: first any subsequent genome alterations to these parental green cells would most probably incorporate a neo (neomycin) selectable marker as it is more widely used than puro (puromycin), and second, the killing efficiency of puro within a short period of time (that conferred during transient drug resistance -since the drug resistance encoding plasmid is electroporated in circular form) is greater than that of neo.
A total of 10 co-electroporations were carried out with the pPGKPuro selection plasmid, and 4 with ploxPneo. Only colonies from the pPGKPuro co-electroporations were picked for expansion and freezing. Differences were observed in the intensity of EGFP
expression in cells in culture. This was first noted when puromycin resistant colonies were assayed for expression of EGFP prior to their picking into 96 well plates.
Identifying ubiquitous expressing green ES cell lines: The initial aim was to establish a line of ES cells that exhibited ubiquitous GFP expression in all embryonic and adult tissues.
Even though the promoter/enhancer combination utilized within the pCX-EGFP
construct has been reported to drive strong uniquitous expression in mice, position effects can often influence the expression of a transgene insertion in a locus specific fashion.
To this end tetraploid aggregations between the puro restistant green ES cells and wild type tetraploid host embryos were performed so as to assay the extent of expression within embryos of several lines. Lines were subsequently chosen for diploid aggregations so as to produce germline transmitting chimeras.
Chimeric analysis to assay fidelity of green cells as a cell autonomous marker: Several of the lines that were believed to exhibit ubiquitous GFP expression within mouse embryos were chosen for diploid aggregations that were not taken to term, thereby permitting the observation of green ES cells in utero and assessing their fidelity as a marker within chimeras, i.e. could green ES cells be discerned from wild type ES cells within a mixed population in low to high level chimeric embryos.
Generation of Fl mice: Four lines (B4, B5, C 1, and D4) exhibited varying degrees of ubiquitous expression in tetraploid aggregation derived embryos, and were thus chosen for the generation of lines of green mice through dipoid aggregations. Of these lines 3 gave germline transmitting chimeras, B4 did not. The BS and D4 ES cell line aggregations each gave one good chimera, and C 1 yielded 2. Male chimeras produced by diploid aggregation were mated with CD 1 or ICR female mice, with the resulting F 1 s being analysed for Mendelian inheritance of the transgene and for the extent of EGFP expression in the pups.
These results indicate that lines BS and C1 contain autosome integrated transgenes (both sexes of F1 pups randomly inherit the transgene), but D4 contains an X-linked transgene insertion (only Fl females inherit the transgene from a male chimera - note also that R1 is a male ES cell line). The first two D4 chimera mated females gave birth to a total of 22 F1 pups, 11 of which were female, transgene activity (indicated by green newborn pups) was solely confined to these females. Over 100 pups have been born, with only the females exhibiting transgene activity.
Interestingly all three lines (B5, C 1 and D4) exhibited almost ubiquitous green expression in tetraploid embryos, though the actual intensity varied dramatically with C 1 expressing at the lowest level, and BS expressing at the highest. Since it was uncertain if high level GFP expressing lines would be deleterious it was decided to use both C1, BS and D4 for the diploid aggregations. Interestingly of the newborn F1 mice BS
exhibited almost ubiquitous green expression, C 1 showed more restricted expression (primarily in the CNS), and D4 females exhibited fluorescence in a mosaic fashion indicative of X-inactivation.
Non-invasive sexing of pre-implantation embryos: The X-linked transgene insertion in the D4 EGFP ES cell line allows the sex-specific selection of individuals at any stage (from 8 cell stage onwards) of development or adult life. This is the first case where a non-invasive marker can be used for sex selection, being particularly amenable to application during pre-implantation stages of development, thereby creating more efficient and cost effective breeding regimes in mice and other mammals.
In an ongoing experiment matings have been set up between the D4 chimera and estrous CD 1/ICR female mice, E3.5 pre-implantation stage embryos have been flushed from the uterine horns and viewed under GFP optics, in total approximately 50%
appear to contain transgene activity. These blastocysts have been separated into green (prospective female) and non-green (prospective male) pools and transfered into foster mothers which are expected to give birth to single sex pups depending on the colour coded pool of the transferred embryos. Additionally preimplantation stage embryos will be subjected to PCR
protocol that can detect the presence of a Y-chromosome and so discern between the sexes, thereby confirming that the non-invasive method for sexing offspring can be carried out at any stage during development.
Example 2 Generating Green Fluorescent Mice by Germline Transmission of Green Fluorescent ES Cells Establishing lines of green fluorescent ES cells. Embryonic stem (ES) cells represent a pluripotent cell type derived from preimplantation stage embryos. They can be propagated in culture, genetically modified and subsequently reintroduced into animals 14,15, Ubiquitously EGFP (Promega Laboratories, Inc. CA) expressing (green fluorescent) ES cell lines were established by co-electroporation of pCX-EGFP16, an EGFP expressing vector, where the EGFP gene is driven by a CMV (cytomegalovirus) immediate early enhancer coupled to the chicken (3-actin promoter and first intron 1 ~, and pPGKPuro 1 g a selectable marker containing vector. Since the majority of targeting cassettes incorporate a neomycin resistance gene, puromycin was chosen as the selectable marker, thereby facilitating a second in vitro modification event if one is required. Of the surviving puromycin resistant colonies only green clones were selected for further investigation. These clones were visualized as green fluorescing colonies under a dissecting microscope equipped with fluorescent optics, and picked into 96 well gelatinized tissue culture plates. Clones were grown in duplicate with one plate being frozen down as a stock. A variation in the intensity and homogeneity of EGFP expression was observed between clones, possibly reflecting the copy number and the effect of site of integration of the transgene. Figure 1 A shows a line of green fluorescent ES cells grown on gelatinized tissue culture plates, and Fig. 1B is a puromycin resistant green fluorescent colony. In most cases maintenance of the green fluorescence was observed in differentiated ES cells (Fig. 1C), whereas embryoid bodies plated on embryonic feeder cells (Fig. 1D) exhibited ubiquitous fluorescence though at varying levels, supporting previous observations that the promoter used in this study is expressed in all nucleated cells but is upregulated in some lineages. Increased fluorescence was consistently observed in endodermal cells situated on the surface of embryoid bodies (Fig. 1D arrows), and suggest that these cells may be equivalent to parietal endodermal cells which were observed to give increased fluorescence over other cell types in transgenic mouse embryos established from this ES cell line.
Selection criteria for ES cell lines - an in vivo test. In order to produce germline transmitting chimeras several different lines of green fluorescent ES cells were tested for developmental potential and ubiquitous embryonic expression by tetraploid embryo-ES cell aggregation 19(see later section). Four of these lines tested by tetraploid embryo-ES cell aggregation were chosen for diploid embryo - ES cell aggregations20 in order to obtain germline transmitting chimeras.
Pre-selecting chimeric embryos for transfer into recipient females.
Aggregating ES cells with diploid eight-cell stage embryos is a simple and inexpensive means of introducing genetic alterations into mice. Circumventing the transfer of non-chimeric embryos after the aggregation is completed makes the technology more efficient and reduces animal space requirements. The B5/EGFP ES cell line now provides a convenient tool to do so. Figure 2 illustrates different ES cell contributions into morula (Fig 2A) and blastocyst (Fig 2B-F) stage embryos obtained after overnight incubation of eight cell stage embryo-B5/EGFP ES cell aggregates. While ES cells will preferentially contribute to the ICM, where they can either represent the entire ICM (Fig 2B) or just a portion of it (Fig 2C, 4D), they will also occasionally be present in the trophoblast (Fig 2E arrow, and 2F), though it is unclear whether this is a real contribution to the trophoblast lineage or whether these cells are trapped in this region of the embryo never to proliferate.
Germline transmission of the BS/EGFP and C1/EGFP transgene - diploid embryo - ES cell aggregation. Reported below are the results of experiments performed with two of the four ES cell lines selected for germline transmission, which were exhibiting extremes in expression of the transgene, as assessed by tetraploid aggregation (these being B5/EGFP a line exhibiting a high level of ubiquitous green fluorescence and C
1/EGFP a line exhibiting ubiquitous green fluorescence at a low level). They were chosen since no data was available on the viability of homozygous mice expressing EGFP at high levels, and to ensure that the integration did not disrupt an essential locus. Figure 3A is a schematic illustration of the diploid aggregation technique in which a single wild type morula stage host embryo is aggregated with a clump of green fluorescent ES cells resulting in the preferential incorporation of the cells into the inner cell mass (ICM) of an embryo by the time it reaches the blastocyst stage (after 24 hours of incubation). The sequence in Figures 3B-E shows the progression of aggregation during overnight incubation. In order to observe the fidelity of EGFP as a cell autonomous marker within a mosaic population of cells, some of the transferred blastocysts were recovered at later (postimplantation) stages of embryonic development. Individual EGFP expressing cells can be identified in chimeric embryos at stages E7.75 (Fig. 4A) and E8.5 (Fig. 4B).
It was also noted that in addition to black eyes and pigmented coat (representative of the 129 mouse strain the Rl ES cells are derived from), chimeras could be identified and assessed for the strength of their ES cell contribution by viewing newborn pups from aggregation recipients under the appropriate fluorescence optics (Fig. 4C and 4D, show a strong chimera). Male chimeras with a high ES cell contribution were crossed with CD 1 or ICR females, and F1 mice were genotyped using the appropriate optics. Both the and C 1/EGFP chimeric mice transmitted the EGFP transgene in a Mendelian fashion.
BSBGFP mice were observed as ubiquitously green throughout embryonic development and adult life (see following section and Fig. 6), whereas in the C1/EGFP line, EGFP was ubiquitously expressed (though at a lower level than in the B5/BGFP line) during early to mid-gestational embryonic development then became restricted to pancreas and certain regions of the CNS in newborns and adults (data not shown). Therefore the data presented here will focus on the B5/EGFP line.
Expression of the BS/EGFP transgene in preimplantation embryos. The onset of zygotic transgene expression was assayed for from the two-cell stage on. No green fluorescence was observed in 1 to 4-cell stage embryos derived from crosses between transgenic males obtained after germline transmission and wild type females (Fig. 5A).
Zygotic expression from the B5/EGFP transgene initiates at preimplantation stages of development, with green fluorescence first detected as morula stage embryos begin to compact (Fig. 5B), and by the blastocyst stage expression is clearly visible (Fig. 5C and 5D).
On the other hand, from the analyses of non-transgenic embryos derived from matings between wild type males and heterozygous females, it was found that residual maternal expression of the transgene is observed until after implantation (Fig. 5E-5G), suggesting that the EGFP transcript or protein is very stable or that the maternal transcript is very abundant.
Due to the high level of maternal transgene expression, all preimplantation stage embryos derived from such a cross were observed as being green fluorescent, though only half the embryos inherited the transgene (Fig 5E-5G). It was also noticed that green fluorescence resulting from maternal expression is more pronounced than zygotic expression up until the blastocyst stage. A comparison of embryos derived from a heterozygous male crossed to a wild type female (Fig. 5H left, note that about half the embryos are green), and a wild type male crossed to a heterozygous female (Fig. 5H right, note that all the embryos are green though only half will carry the EGFP transgene) illustrates this observation.
The residual maternal transgene expression observed in preimplantation embryos is lost after implantation, probably as a result of the degradation of the EGFP transcripts or protein, and the rapid increase of the cellular mass that is observed concurrent with implantation at E4.5-4.75, and the onset of gastrulation at E6.5.
Expression of the B5/EGFP transgene in postimplantation embryos.
Postimplantation expression of the BS/EGFP transgene in embryos and extraembryonic tissues appeared to be ubiquitous throughout development (Fig. 6). It was also found that the green fluorescence facilitated the location and isolation of postimplantation embryos at earlier postimplantation stages (E4.75-E5.75) which is otherwise extremely difficult. This means that such a green fluorescent transgenic line allows access to embryos at all stages of development; from preimplantation through postimplantation to birth. Fig. 6A
shows an E4.75 (late hatched blastocyst) green fluorescent embryo derived from a cross between a B5/EGFP male and a wild type female, the embryo has just implanted and is surrounded by some residual non-fluorescent uterine tissue. Fig. 6B shows a uterus that has been cut open at the proximal side (opposite the mesometrial side) showing early stage decidual tissue which has been torn open to expose an ES green fluorescent embryo. Figure 6C
shows an E5.5 green fluorescent embryo dissected away from the uterus but still surrounded by some residual decidual tissue. By E9 (Fig. 6D, 6E) it is clearly visible that both the embryo proper and extraembryonic tissues express the EGFP transgene. Starting at E9 in the extraembryonic lineage (Fig. 6E), the parietal endoderm exhibits highest levels of transgene expression, this observation is supported by the differentiated embryoid body data where endoderm cells regularly exhibit increased fluorescence (Fig. 1D arrows). At all postimplantation stages no fluorescence is observed in non-transgenic embryos, as illustrated in Figures 6F and 6G. Figure 6F shows a bright field normal optic view of two E9.5 littermates derived from a cross between a transgenic male and a wild type female (only one has inherited the transgene), with Fig. 6G being a dark field fluorescent optic view where only the transgenic embryo can be seen to fluoresce. The residual maternal transgene expression observed in preimplantation embryos is thus lost after implantation, probably as a result of the rapid cellular proliferation that is observed concurrent with implantation at E4.5-4.75, and the onset of gastrulation at E6.5. After midgestation transgene expression is maintained in both the embryo and placenta (Fig. 6G). All tissues appear to express the transgene including the heart and lungs (Fig. 6I) in addition to the kidney rudiment and intestine (Fig. 6J). The only cells that appear not to fluoresce green are enucleated cells such as erythrocytes. Additionally cells and tissues with increased hemoglobin content, such as blood vessels, spleen and liver, exhibit reduced fluorescence as their development proceeds since the hemoglobin masks the green fluorescence.
Expression of the BS/EGFP transgene in adult mice. As with transgenic embryos, the BS/EGFP transgene is ubiquitously expressed in newborn (Fig. 7A-7C) and adult mice (Fig. 7D-7F). Transgenic newborn pups can be recognized due to fluorescence in the skin (Fig. 7A, 7B), but as pups mature the fluorescence is obscured in the fur coat covered body parts, especially if the fur is pigmented (Fig. 7C). This contrast is clearly observed between adult mice with different coat colours, and is illustrated here by albino (Fig. 7D and 7E), grey (chinchilla), and agouti (Fig. 7F, tails left to right). The entire organ system of the BS/EGFP transgenic line fluoresces green and, even though ubiquitous, the level of expression varies between different organs, with musculature (Fig. 7G), brain (Fig. 7H) and pancreas (Fig. 7K) exhibiting the highest levels of transgene expression, intestine (Fig. 7L) expresses at a lower level. Kidney (Fig. 7I) and liver (Fig. 7J) also do not strongly fluoresce as they have a high hemoglobin content.
Mouse embryos derived solely from ES cells: wt tetraploid embryo - GFP ES
cell and GFP tetraploid embryo - wt ES cell aggregations. The aggregation of ES cells with tetraploid 4-cell stage embryos results in the production of a completely ES cell derived postimplantation embryo, and provides a way to study the phenotype of a given ES cell line, circumventing germline transmission19,21,22~ or to separate embryonic and extraembryonic components of a compound phenotype resulting from gene ablation23. The technique is illustrated in Figure 8A, with Figures 8B-E illustrating the process of aggregation. The top panel of Figure 9 shows embryos derived from a wild type tetraploid embryo -BS/EGFP ES
cell aggregation experiment where the embryo will be green fluorescent (Fig.
9A, 9B), whereas the lower panel illustrates the reverse a BS/EGFP - wild-type ES cell aggregation where the ectoplacental cone/placenta will be green fluorescent (Fig. 9C, 9D).
With simple microscopic observation the expected separation of the tetraploid and ES cell compartments to the extra embryonic tissues and embryo proper, respectively, could be confirmed.
Discussion The BS/EGFP line of ES cells represents a source of pluripotent, green fluorescent cells which can be manipulated in vitro by further transgenesis or targeted gene alteration, then reintroduced into mice. Germline transmission of these ES cells has allowed us to establish that EGFP is ubiquitously expressed in all cellular progeny of the ES cells. Thereby EGFP would provide a mutant cell-autonomous marker for mutant cells which would then be coupled to a second genetic alteration. These cells could be used for mutant cell transplantation experiments or chimeric analyses, where the GFP expression provides an exceptional tool to follow the behavior of the mutant cells during development or disease processes, such as cancer.
Alternatively, the transgenic mouse line derived from the "primary" BS/EGFP ES
cell line can provide a ubiquitous GFP tag for normal cells and tissues for similar experiments where opposite marking is required. In addition, green fluorescence can be used as a means to identify and dissect early stage preimplantation embryos at stages (E4.75 - 6.0) that have previously been elusive to all but the most highly skilled researchers. Beside the above main applications other important uses are envisioned for the GFP tagged ES-cell /
transgenic-mouse system. Green fluorescent ES cells should increase the efficiency of chimera production by allowing the preselection of chimeric blastocysts prior to transfer into recipient females.
Unlike standard transgenic regimes where genotyping is performed by either PCR, Southern analysis or the use of a chromogenic substrate for reporter gene visualization, using the present invention transgenic embryos and pups were identified by visualization of green fluorescence. When a litter of pups derived from a cross between an animal heterozygous for the transgene and a non-transgenic is viewed under the appropriate optics only half the animals will fluoresce (e.g. Fig. 10). Such a fluorescence based reporter strategy offers the advantage not only in that transgenic animals are readily and non-invasively identified, but also that animals homozygous for the transgene can be recognized due to their increased fluorescence over heterozygotes a feature that can only be determined in standard transgenics by breeding animals or by cloning the site of transgene insertion.
The tetraploid embryo - ES cell aggregation provides a fast and inexpensive access to the embryonic phenotypes of mutations created in ES cells and also generates polarized chimeras in which the embryo proper, amnion and yolk sac mesoderm is compeletely ES

cell-derived whereas the yolk sac endoderm and trophoblast lineages are tetraploid embryo-derived (Nagy, A et al, Development 110, 815, 1990). The feasibility of this technology was demonstrated in an earlier study2g. In such studies it is essential to tag the tetraploid cells with a reporter gene to ensure that the embryos are completely mutant ES cell-derived. Until now, the Rosa 26 transgenic line24 providing ubiquitous expression of a lacZ
reporter has been the line of choice. Here the superiority of the GFP transgenic mouse line is demonstrated for tagging the tetraploid compartment of an embryo.
Through demonstrating some of the applications of the ubiquitous GFP reporter system in ES cells and the corresponding transgenic mouse line, the intensively evolving mouse genetic technologies will incorporate the tool described herein in many aspects of its methodologies.
Experimental protocol Generation of green ES cells. pCX-EGFP, an EGFP containing vector has been described previous1y10. It contains EGFP inserted into pCAGGS, a plasmid vector containing a chicken beta-actin promoter and CMV enhancer, Beta-actin intron and bovine polyadenylation signally. pPGKPuro, a puromycin resistance gene containing vector containing a PGK promoterl8 was used for co-electroporation since it carries an antibiotic resistance.
R 1 ES cells 19 were maintained as described previously25. Cells were co-electroporated under standard conditions25,26 with 40-80p,g of linearised pCX-EGFP
(BamHI or ScaI were used interchangeably for linearisation) and lOmg of circular pPGKPurolB. Each individual electroporation was seeded onto a single lOmm gelatin coated tissue culture plate and resulted in approximately 80-140 puromycin resistant colonies, 8-10 of which were green as observed under the appropriate optics. Control electroporations were carried out using one or other of the plasmids singly with subsequent selection in puromycin.
Selection in puromycin was initiated 36-48 hours after electroporation, and continued for 3-4 days thereafter. Colonies were viewed under a dissecting microscope (Leitz MZ8) equipped with a GFP illumination source (Leica) and scored for green by etching the underside of the tissue culture plate with a needle. Colonies assigned as being green were picked into 96 well plates for expansion and freezing25.
Embryoid bodies were obtained by seeding ES cells into bacteriological plates and by omitting LIF and (3-mercaptoethanol from the culture medium. After approximately 1 week these embryoid bodies which had been growing in suspension were replated onto feeder cell containing plates, where they attached and differentiated further2~.
Diploid embryo - ES cell aggregations. Diploid aggregations were performed as described previous1y20. A detailed protocol can be obtained on the world wide web on http://www.mshri.on.ca/develop/nagy/Diploid/diploid.htm. After overnight incubation with cells in depression plates embryos were either transferred to recipient females (for the production of germline transmitting chimeras or postimplantation embryo dissections), seeded onto feeder cell containing plates (for the analysis of blastocyst outgrowths) or placed in M2 medium in depression glass microscope slides (for photography).
Germline transmitting male chimeras were crossed with ICR females.
Subsequently, the BS/BGFP mice have been maintained on an ICR background.
Tetraploid embryo - ES cell aggregations. The technique relies on the electrofusion of 2-cell stage embryos placed between the electrodes of an electrode chamber slide, resulting in rendering their genome tetraploid. The resulting one-cell stage embryos are incubated in vitro until they reach the 4-cell stage of development at which time they are used to 'sandwich' a clump of ES cells. This arrangement when incubated overnight promotes the aggregation of the two embryos and the intervening ES cells resulting in the formation of a blastocyst. Tetraploid aggregations were performed as described previously2l. A detailed protocol can be obtained on the world wide web on http://www.mshri.on.ca/develop/nagy/Tetraploid/Tetra.htm. After aggregation embryos, were treated exactly as for diploid aggregations.
Observation of green fluorescence. Oviducts or uteruses of superovulated females were flushed for recovery of preimplantation stage embryos, PBS was used if embryos were not to be used for in vitro culture, otherwise M2 or KSOM media was usedl.
Postimplantation embryos were dissected in PBS and viewed under both dark field and bright field light optics. Certain types of plasticware were found to be refractory to fluorescence, and glass vessels and microscope slides gave superior clarity.
For plasticware Costar lOcm tissue culture, Fisher lOcm bacteriological, and Falcon 3cm tissue culture and organ culture were routinely used for viewing cells and embryos under fluorescent optics.
For long-term storage and sectioning, embryos and adult tissues were fixed in 4°Io paraformaldehyde at 4°C for between 30 min to 4 h. Fifty p,m sections were cut in ice-cold PBS using a vibratome. Sections were mounted under coverslips in l:l glycerol/PBS and were stored at 4°C.
EGFP fluorescence was examined under dissecting microscopes (Leitz MZ8 and MZ12) or inverted microscopes (Leitz DMRB or DMRXE) equipped with GFP
excitation sources and appropriate filters (Leica GFP Plus fluorescence filter set).
Photographs of fluorescent subjects were taken on Kodak p1600 film shot at 800 or 1600 ASA.
Mice were routinely genotyped without magnification using a Volpi fibre-optic light source fitted with a blue excitation filter and viewed through a yellow (520nm) barner filter (Chroma).
Photographs of pups and mothers were taken using a Nikon FE10 SOmm camera fitted with a 35-75mm lens, a set of close-up filters, and a yellow (520nm) barrier filter.
Example 3 Non-invasive determination of sex prior to morphological gonadal differentiation has long been sought, but up until now no such assay has been available for widespread use. For example in mammals, the embryo develops to a substantially advanced stage within the mother, thereby requiring the maternal investment of time and energy.
Therefore from many viewpoints information regarding sex outcome could be beneficiall if determined at preimplantation, prior to the establishment of the pregnancy. Several attempts have been made to identify or predetermine the sex of animals at an early stage of embryogenesis, including density centrifugation-separation of sperm into Y and X chromosome bearing populations, followed by in vitro fertilization2, specific elimination of preimplantation stage male embryos by antibodies directed against a Y chromosome-specific surface antigen3, measurement of X-linked gene dosage between littermates4, in addition to bar body staining5, karyotyping6 and PCR detection for the presence of a Y chromosomes using biopsy samples taken from preimplantation stage embryos. Each of these methods is invasive, labor intensive, potentially harmful to the embryo and subject to erroneous results.
GFP is a protein of the jelly fish Aqueoria victoria whose marking of individual cells in a heterologous system was first demonstrated in the nematode worm C.
elegans 8. It is now becoming the reporter of choice in many different systems, from simple organisms such as yeast to vertebrates9. GFP acts as a reporter of gene expression that can be viewed in a non-invasive manner, since it can be visualized without the use of a substrate by simple observation under specific illumination. At present GFP and its mutagenised derivativesl0 provide the only non-invasive markers of gene expression available for use in any biological system.
In the process of establishing green fluorescent ES cell lines in order to create transgenic mice ubiquitously expressing this novel reporter, a mouse transgenic line was generated carrying an EGFP (Clontech Laboratoties, Inc.) transgene integrated on the X
chromosome (D4/XEGFP transgene). This line represents the first vertebrate system where sex can non-invasively be determined from approximately 48 hours after fertilization, long before any of the morphological landmarks that discriminate between the sexes have been established.
Over 500 pups have been born from fathers carrying the EGFP marked X
chromosome, and in all cases transgene activity in the F1 generation was solely confined to females (Fig. lla). F1 females (hemizygous for the transgene) when crossed to non-transgenic males produce offspring 50% of which harbor the transgene in a non-sex specific manner. Transgenic male offspring derived from such a mating, between a transgenic female and wild type male, ubiquitously express the transgene in the skin, whereas hemizygous females exhibit mosaic expression due to random inactivation of one of their two X
chromosomes (Fig. llb). If a transgenic male is crossed to a hemizygous female 75% of progeny carry the transgene, represented by three distinct populations;
transgenic males exhibiting ubiquitous green fluorescence (25% of offspring), hemizygous females exhibiting mosaic green fluorescence (25% of offspring), and homozygous females exhibiting ubiquitous green fluorescence (25% of offspring).
No discernable phenotype, other than green fluorescence, has been observed in mice carrying the D4/XEGFP transgene in either hemi- or homozygous form.
Interestingly the banded pattern of green fluorescence, rarely seen to cross the dorsal or ventral midline, observed in the skin of newborn hemizygous female pups, appears to be intriguingly similar to the fur coat coloring mosaicism characteristic of a variety of other animals including tabby cats, and mutations at the X-linked Mottled (Mo) locus in micel 1.
The extent of the D4/XEGFP transgene expression has been determined during embryogenesis. Zygotic expression of the transgene first produces a detectable green fluorescence in all cells of the embryo initiating at approximately embryonic day (E) 2.5 2.75, as morula stage preimplantion embryos begin to compact. Studies on postimplantation embryos, newborn pups and adult mice reveal that the transgene is expressed in all nucleated cell types catalogued, with expression being slightly higher in heart than in other tissues (Fig. 12b-c, and Fig. 14d). Additionally in hemizygous females where the transgene is randomly inactivated in approximately 50°70 of all cells of the embryo proper (starting at E6.5-7.0 around the time of gastrulation), close inspection of embryos and fluorescence activated cell sorting (FAGS) analysis of dissociated cells from such embryos suggests that transgene expression is mosaic.
In order to further compound the idea that preimplantation stage embryo could be non-invasively sexed, it was decided to color pool, and therefore single sex pool, preimplantation stage embryos and then to transfer them to recipient surrogate females. If the color coded non-invasive sexing was truly representative, then these females would be expected to give birth to single sex litters, of which the female only would fluoresce green.
Matings were set up between D4/X-EGFP transgenic males and wild type female mice. Pre-implantation stage embryos were flushed from the uterine horns or oviducts of pregnant females, and viewed under the appropriate illumination for GFP
expression in order to discern their sex. Embryos were then separated into green fluorescent (prospective female) and non-green fluorescent (prospective male) pools, and transferred to pseudopregnant surrogate females (Fig. 13). Approximately 200 preimplantation embryos were color pooled and transferred in this way, encouragingly no deviations from the expected single sex litters were observed.
Preimplantation and midgestation stage embryos were also subjected to PCR
sexing, in order to detect the presence of a Y chromosome, and so discern between the sexes (Fig.
14). This PCR based approach is currently the favored protocol used for preimplantation sexing of mammalian embryos. The results indicate that only non-fluorescent embryos show evidence for the presence of a Y chromosome. This invasive result further supports the observations that the X-linked EGFP transgenic line offers a non-invasive method for sexing offspring at any stage during embryonic development.
The D4/XEGFP transgenic line is the first reported case where a non-invasive marker has been used for sex selection in any organism before sexual dimorphism makes the discrimination possible. Such an approach is feasible, and particularly amenable to application during pre-implantation stages of development, thereby creating more efficient and cost effective breeding regimes in human controlled sexually reproducing species. In the case of mice a significant impact of such a preimplantation sex selection on ES cell based genetic technology is envisioned. By selecting female host embryos for injecting or aggregating with male ES cells, 100% germline transmission of the ES cell genome is guaranteed in fertile male chimeras, since only XY containing cells (ES cells in this case) can undergo spermiogenesis. In larger mammals such as cattle, where breeding is expensive and time consuming, costs can be streamlined by such non-invasive preimplantation sex selection.
Methods Details of vectors used. R 1 ES cells 12 were co-electroporated under standard conditions 13 with 40-80~g of a linearised EGFP expressing plasmid pCX-EGFP14 and lOmg of a circular antibiotic resistance carrying plasmid, pPGKPurols. Individual electroporations were seeded onto lOcm gelatin coated tissue culture plates. After the appropriate period of antibiotic selection 80-140 drug resistant colonies remained, approximately 5% of which were green.
Cells were allowed to recover for 2 days prior to initiation of the drug selection.
Production of transgenic mice. Diploid aggregations 16 were used to transmit the D4/XEGFP transgene to the germline. The protocol currently used is available on the world wide web on http://www.mshri.on.ca/develop/nagy/Diploid/diploid.htm.
Collection and color coded pooling of preimplantation stage embryos. Matings were set up between D4/XEGFP transgenic male mice and wild type ICR or CD 1 females.
Noon on the day of plug formation was taken as representing E 0.5. At E2.5 oviduct, and at E3.5 uteruses were flushed with M2 medium. Eight cell stage embryos or blastocysts were collected and assayed for green fluorescence. Embryos were pooled into green and non-green groups then either used for individual PCR genotyping, or transferred directly to recipient pseudopregnant females using standard proceduresl~.
Observation of EGFP activity. GFP fluorescence was examined under dissecting microscopes (Leitz MZ8 and MZ12) equipped with GFP excitation sources (Leica GFP
Plus). Both ES cells and mouse embryos were viewed using the same optics.
Newborn pups were observed and photographed on the stage of a dissecting microscope from which the condenser lens had been removed. They were viewed through a yellow burner filter (Chroma) which was mounted onto a Nikon FE10 35mm camera, or incorporated into a pair of eyeglasses.
PCR analysis. PCR genotyping was carried out on individual preimplantation stage embryos and yolk sacs from postimplantation stage embryos. The presence of a Y
chromosome was assayed for using primers for Sry 18 and the presence of an autosomal gene (positive control) was assayed using primers for myogenin 19. For postimplantation embryos 30 cycles were routinely used, whereas for preimplantation stages two rounds of PCR of 25 cycles each were used. Annealing temperature was 60°C.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

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Example 2:
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Figure Legends:
Figure 1.
Expression of EGFP in undifferentiated and differentiated ES cells. (A) ES
cells and (B) drug resistant colonies, exhibiting ubiquitous EGFP expression. (C) differentiated ES
cells and (D) embryoid bodies also exhibit ubiquitous transgene activity, though the latter do show differential levels of expression with the surface endoderm layer often containing a population of highly fluorescent cells (indicated by an arrow).
Figure 2.
Preselection of embryos with a good ICM contribution for transfer into recipient females. (A) ES cells colonizing part of (right) and the majority of (left) morula stage embryos. (B) ES cells colonizing most of the ICM but not the trophectoderm of a blastocyst stage embryo. (C,D) ES cells colonizing a proportion of the inner cell mass (ICM) of a blastocyst. (E) ES cells localized to the majority of the ICM with a single cell (arrowed) in the trophectoderm (tro) of a blastocyst, and (F) ES cells localized to part of the ICM with two cells seen in the trophectoderm.
Figure 3.
Diploid green ES cell - wild type embryo aggregations. (A) schematic diagram detailing the technique. (B 1-B3) aggregation of a clump of green fluorescent ES cells with a wild type embryo results in blastocyst formation after overnight incubation, with preferential contribution of the ES cells to the inner cell mass (B4).
Figure 4.
Postimplantation embryos made by diploid embryo - ES cell aggregations.
Embryos exhibit mosaic expression of EGFP reflecting their chimerism between wild type (non-fluorescing) and BSBGFP ES cell contributions. (A) ventral view of a late headfold/presomite stage (E8) embryo, anterior is top, (B) lateral view of an early somite stage (E8.5) embryo, anterior is left, (C) dorsal view of an E8.5 embryo, anterior is left, (D) lateral view of the head and forelimb of a newborn chimera, (E) view of the hindlimbs and tail of a newborn chimera, anterior is up.

Figure 5.
BS/EGFP expression in preimplantation embryos. All embryos in panels A-D are derived from a cross between a male that is heterozygous for the transgene and wild type females, whereas embryos in panels E-H are derived from a cross between a wild type male and a female heterozygous for the transgene. (A) 1 - 4-cell stage embryos exhibiting no transgene expression, (B) embryos initiate expression at the morula stage (~E2.5). By the blastocyst stage expression of the transgene is clearly visible, (C) dark field and (D) bright field view of E3.5 embryos. Residual transgene expression due to maternal transcripts is observed until implantation stages, 2-cell and morula stage green fluorescent embryos bright field (E), dark field (F), dark field of blastocyst stage embryos (G). At E2.5 fluorescence due to maternal transcripts (right) is stronger than zygotic (left).
Figure 6.
B5/EGFP expression in postimplantation embryos. All embryos derived from a B5 male crossed with ICR female. (A) E4.75 newly hatched and implanted blastocyst attached to decidual tissue (dec), (B) E5, (C) E5.5, (D) E9 embryo with associated ectoplacental cone (EPC) partially contained within the deciduum, (E) E9 extraembryonic membranes, (F), bright field view of two E9.5 littermates, (G), dark field view of the same two embryos, (H) E11.5 embryo with placenta, (I) E11.5 heart and lungs, (J) E11.5 kidney rudiment and intestine.
Figure 7.
Ubiquitous green fluorescence in B5/EGFP mice and organs. (A) head and forelimb view of a newborn B5/EGFP heterozygote pup among non-transgenic littermates, head transgenic right and non-transgenic literate left, (B) newborn B5/EGFP heterozygote transgenic (right) and non-transgenic literate (left), (C) 1 week old pups transgenic on the right non-transgenic literate left, (D) 3 week old albino B5/BGFP mouse anterior view, (E) 3 week old albino B/EGFP mouse posterior view, (F) posterior view highlighting the tails of two month old albino (left), grey (middle), agouti (right) mice illustrating pigment dependent fluorescence, (G) dorsal view of an incision made in 3 week old B5/BGFP mouse body with skin removed to reveal fluorescence in the body. Organs from a 3 week old transgenic mouse right/top and non-transgenic littermate left/bottom; (H) brain, (I) kidney, (J) liver, (K) pancreas, (L) intestine.

Figure 8.
Tetraploid aggregations to produce completely BS ES cell derived embryos. (A) schematic diagram detailing the tetraploid technique. (B-E) the progression of an aggregation between green ES cells <-> wild type embryo to the blastocyst stage.
Figure 9.
Postimplantation embryos made by tetraploid aggregation. (A,B) embryos made by BS
ES cell - wild type tetraploid embryo aggregation. (C,D) embryos made by wild type ES cell - BS embryo aggregation. (A) green fluorescent E9 embryo with non-fluorescent extraembryonic membranes (outline by a hatched line), (B) green fluorescent E11 embryo with mosaic yolk sac and non-fluorescent placenta, (C) E8.5 embryo with green fluorescent extraembryonic membranes, (D) E11 embryo with mosaic yolk sac and fluorescent placenta.
Figure 10.
Non-invasive genotyping of embryos and adults by U.V. - Mendelian inheritance of the BS/EGFP transgene. Genotyping heterozygotes vs non-transgenic animals: (A) newborn pups from a cross between a BS/EGFP heterozygous male and an ICR female, between every fluorescent (transgenic) pup is a non-fluorescent non-transgenic animal (indicated by an asterix). Genotyping heterozygotes vs homozygotes: (B) mice from a litter of pups derived from an intercross between a male and female both of which where heterozygous for the transgene (homozygotes are highlighted by an asterix), (C) a close view of pups heterozygous (left) and homozygous (right) for the transgene, (D) tail tips from E11.5 embryos homozygous (top), heterozygous (middle) and non-transgenic (bottom).
Figure 11. Sex-specific GFP expression in newborn pups. A litter of F1 offspring (males left, females right) derived from a cross between a D4/XEGFP transgenic male and a wild type female (a). 6 pups from a litter derived from a cross between a hemizygous female and wild type male (b). Three phenotypes are observed, non-transgenics that do not show any green fluorescence (male in the first cross, male and female from the second cross), hemizygous females that exhibit mosaic fluorescence in the skin, and transgenic males that exhibit homogenous fluorescence. Homozygous females though not shown, are phenotypically identical to transgenic males in being homogeneously fluorescencent.

Figure 12. Expression of the D4/XEGFP transgene in a sex-specific manner in postimplantation stage embryos prior to sexual morphogenesis. F1 embryos at E6.5 (a), E8.5 (b), and E10.5 (c), derived from matings between transgenic males and wild type females are shown. Non-transgenic males, on the left, can clearly be discerned from hemizygous females, on the right. Close visual inspection of the female embryos, or analysis of dissociated cells from them reveals that the observed green fluorescence is in fact mosaic.
Figure 13. Preimplantation stage transgene expression allows sex-specific pooling. A pool of three litters of E3.5 embryos derived from natural matings between transgenic males and wild type females in bright field (a), dark field with background illumination (b), and full dark field (c), where green fluorescent embryos are female and the non-flluorescent embryos are male (b). Color and sex pooled embryos (d), which gave rise to single sex litters after transfer to surrogate females.
Figure 14. Invasive PCR sexing of preimplantation, and midgestation embryos.
Bright field (a) and dark field view (b) of a litter of E3.5 preimplantation stage embryos obtained from a mating between a D4/XEGFP transgenic male and a superovulated wild type female. A
dark field view (with background illumination) of a litter of E10 embryos obtained from a mating between a D4/XEGFP transgenic male and a wild type female (c). PCR
genotyping the E3.5 (d) and E10 (e) embryos for Sry and myogenin.

Claims

1. A pluripotent cell comprising a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into a heterologous sex chromosome in the cell.

2. A pluripotent cell line comprising a pluripotent cell as claimed in claim 1.

3. A pluripotent cell as claimed in claim 1 which is an embryonic stem cell.

4. A pluripotent cell as claimed in claim 1 wherein the fluorescent protein marker is green fluorescent protein or a variant thereof.

5. A chimeric embryo comprising a pluripotent cell as claimed in claim 1.

6. A method for producing a pluripotent cell that has a heterologous sex chromosome linked fluorescent protein marker comprising introducing into a pluripotent cell a nucleic acid sequence encoding a fluorescent protein marker, and optionally a nucleic sequence encoding a protein of interest, under conditions so that the fluorescent protein marker is selectively integrated into a heterologous sex chromosome in the cell.

7. A method as claimed in claim 6 wherein the pluripotent cell is an embryonic stem cell, and the heterologous sex chromosome is an X chromosome.

8. A method as claimed in claim 7 wherein the fluorescent protein marker is green fluorescent protein or a variant thereof.

9. A method of producing a chimeric embryo that develops into a non-human animal that transmits to its progeny a nucleic acid sequence encoding a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in the animal's cells comprising:
(a) providing pluripotent cells as claimed in claim 1; and (b) introducing the pluripotent cells in an embryo or aggregating the pluripotent cells with an embryo to produce a chimeric embryo.

10. A method as claimed in claim 9 further comprising (c) implanting the chimeric embryo into a pseudopregnant foster mother, and (d) allowing the foster mother to grow to term to produce a non-human transgenic animal that is capable of transmitting the nucleic acid sequence only to female or male progeny.

11. A non-human transgenic animal having a fluorescent protein marker selectively integrated into one of the heterologous sex chromosomes in its cells produced by a method as claimed in claim 10.

12. A method for producing a non-human animal having a fluorescent protein marker integrated into one of the heterologous sex chromosomes in its cells comprising;
(a) allowing an embryo as claimed in claim 5 to develop to term to produce a non-human animal; and (b) optionally breeding the non-human animal and selecting progeny that express the fluorescent protein marker.

13. A method as claimed in claim 12 wherein the non-human animal is a rodent.

14. A method of monitoring expression of a protein of interest linked to a heterologous sex chromosome in a non-human animal comprising:
(a) introducing into pluripotent cells a transgene comprising a DNA sequence encoding a protein of interest and a sequence encoding a fluorescent protein marker under conditions so that the fluorescent protein marker is selectively integrated into a heterologous sex chromosome in the cells;
(b) culturing the pluripotent cells in conditions permitting expression of the fluorescent marker protein and the gene of interest; wherein the pluripotent cells expressing the fluorescent marker protein express the gene of interest; and (c) monitoring the expression of the gene by monitoring expression of the fluorescent protein marker in the pluripotent cells.

15. A method as claimed in claim 14 further comprising injecting the pluripotent cells into a blastocyst or aggregating them with an early stage embryo to produce a chimeric embryo, and monitoring the expression of the protein by expression of the fluorescent protein marker in the chimera or in the growing chimera.

16. A method as claimed in claim 14 wherein the fluorescent protein marker is green fluorescent protein or a variant thereof.

17. A method as claimed in claim 15 further comprising implanting the chimera into a pseudopregnant foster animal and monitoring the expression of the gene by monitoring expression of the fluorescent protein marker in the growing chimera or in the resultant transgenic animal.

18. A method as claimed in claim 14 wherein the pluripotent cells are embryonic stem cells.

19. A method of selecting male or female embryos comprising: producing embryos containing a fluorescent protein marker selectively integrated into a preselected heterologous sex chromosome in accordance with a method as claimed in claim 9;
and selecting male or female embryos depending on whether the embryos expresses the fluorescent protein marker.

20. A method as claimed in claim 19 wherein the preselected heterologous sex chromosome is an X-chromosome.