NZ207393A - Staining dna in living cells - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £07393 2073 9 NZ PAT"\T OFFICE -6 MAR 1984 RSCEivnO _ _ NEW ZEALAND KILDBAW3KSS „act.1953 No.: Dale: COMPLETE SPECIFICATION "TECHNIQUE FOR VISUALIZATION OF GENETIC MATERIAL" X/ We, NEAL LLOYD FIRST, of 1717 Heim Avenue, Madison, Wisconsin 53706, U.S.A. ; ELIZABETH SHEA CRITSER, of 310 N. Westfield Road, Madison, Wisconsin 53717, U.S.A.; and JAN KARLOTTA LOHSE, of 610 Ozark Terrace, Madison, Wisconsin 53706, U.S.A., all are citizens of the United States of America, hereby declare the invention for which ^ / we pray that a patent maybe granted toxxse</us, and the method by which it is to be performed, to be particularly described in and by the following statement: - Priority Date(s): Complete Specification Filed: .4.

Class: Publication Date: 5Vl.ttAB.lS87...

P.O. Journal, No: . ■ /. 2073 BACKGROUND OF THE INVENTION This invention relates generally to staining techniques for biological genetic material. More specifically, the method of this invention provides a unique means of visualizing (1) the pronuclei of living pronuclear cells, including ova and one-cell fertilized eggs (2) the condensed DNA in the heads of spermatozoa, and (3) the nuclei of pre-implantation embryos. Visualization of the genetic material in these types of living cells is critical for further research and development of in vitro fertilization and maturation, gene and nuclear transfer capabilities and subsequent cloning of domestic animal embryos. Heretofore, pronuclei and sperm DNA have not been seen in living, viable mammalian cells, except for rodents and humans where the cytoplasm of these cells is clear- In more mature embryos, the area of the nuclei may be somewhat discernable, but the cells' smaller size makes it impossible to visualize individual nuclei.

Advanced genetic improvement and selection techniques continue to be sought in the field of animal husbandry. With specific reference to dairy cattle, for example, significant increases in milk production have been seen with the wide scale use of genetically superior sires and artificial insemination. Dairy cows today produce nearly twice as much milk as they did 30 years ago. Further genetic improvement can be accomplished by the multiplication of superior or genetically manipulated embryos by cloning. Embryos may be cloned by (1) replacement of pronuclei or nuclei in a fertilized egg cell with donor pronuclei or with nuclei from an early multicellular embryo or (2) transfer of a valuable gene into the pronucleus or nucleus. 2073 9 3 Gene transfer requires the pronucleus or nucleus of the recipient cell to be visible so that DNA fragments containing the desired gene sequences can be transferred by micromanipulation directly into one of the pronuclei or the nucleus. Nuclear transfer, on the other hand, requires the nucleus or the male and female pronuclei to be visible so that they can be removed from the fertilized cell in exchange for a nucleus from the desired clonal line. Techniques such as these involving the manipulation of genetic material were developed in research with murine embryos in which the cytoplasm of the pronuclear and embryo cell is clear, permitting visualization of the nuclear material in living cells by light microscopy.

The situation in bovine and porcine gametes and pre-implantation embryo cells is quite different. The cytoplasm of these cells is dense and granular and contains thick lipid droplets. The genetic material is obscured and cannot be visualized by light microscopy. Conventional nuclear stains are not vital stains; that is, they require that the cells be fixed and cleared prior to staining. Bovine and porcine gametes and pre-implantation embryo cells therefore normally have been examined in fixed, cleared and nonliving specimens.

As discussed above, genetic improvement techniques such as gene and nuclear transfer require the selective staining of the nuclear or pronuclear material in living pronuclear or embryo cells. These techniques, together with a host of laboratory research tasks, are made possible by the technique of this invention which provides a visualization method with the necessary selectivity and which can be used with living cells. 207393 SUMMARY OF THE INVENTION The technique described herein enables the male and female pronuclei to be visualized in living mammalian cells, even in those species in which the density of the cell cytoplasm normally obscures the pronuclei. Visualization of pronuclei in living cells has not previously been reported, except rodent and human cells. The technique also provides a means for visualization of the nuclear material of multi-cell embryos, making it possible to count the number of cells present in a developing embryo after each cleavage as well as manipulate the genetic material for gene and nuclear transfer.

Visualization is accomplished by exposure of the living cells to a stain or dye which is specific for double stranded DNA but which does not preclude the continued viability of the cell. Fluorescent stains, such as DAPI or the bis-benzimidazole compounds, are used. The stain is added to the culture medium containing gametes, one-cell fertilized eggs or multi-cell embryos. After incubation, which can be for as short a time as several minutes, the genetic material can be visualized by exposure to ultraviolet light. The pronuclear or nuclear structures appear as stained areas within the cytoplasm. The intensity varies from very faint to bright fluorescing.

An overall object of this invention is to enable and facilitate laboratory research tasks such as gene and nuclear transfer techniques, and studies of the patterns and progress of i_n vitro maturation of oocytes, and in vitro fertilization and embryo culture. o 2073 93 One of the principal objects of this invention is to provide a technique for the visualization of the pronuclei of living mammalian pronuclear cells.

It is a more specific object to permit visualization 5 of the male and female DNA or pronuclei of living mammalian gametes and one-cell fertilized eggs, bovine and porcine in particular.

It is a further object to provide a like technique which will allow for the visulization of nuclear material 10 of multi-cell embryos.

A related object is to provide a means for the differential staining of the genetic material and the cytoplasmic contents.

Still another object is to provide a means of staining 15 the genetic material in living germ and embryo cells in a manner which is likely to allow maintenance of the health and viability of the Cells.

In addition, it is desired to provide a stain which will not cause excessive damage or alteration to the 20 genetic material.

DETAILED DESCRIPTION OF THE INVENTION The method of this invention provides a technique for the differential staining of certain genetic material which never before has been visible in living cells. The 25 fluorescent stains used in this method selectively stain the genetic material of the cells, without requiring the cells to be fixed and cleared. The selectivity of this staining technique provides a powerful research tool which will facilitate further investigation of fertilization, 30 embryological development and gene and nuclear transfer in domestic animal embryos. 2073 The stains of this method can be used to selectively stain the genetic material of any species. However, the technique finds its highest value with those species in which the cytoplasmic materials (e.g., lipids) obscure the pronuclear or nuclear material in the living state. Examples of considerable interest and importance to the animal breeding industry are cattle and swine.

Application of the technique to other research or domestic animals is contemplated as well.

It has been found that certain fluorescent stains used according to this method will achieve the dual objectives of selectivity of staining and continued viability of the cells. Fluorescent stains which are so-called "vital stains," that is, which do not require the cells to be fixed and cleared and which do not interfere with the continued development of the cell, may be useful in this procedure. In addition, the stain should be of the type which is incorporated into the cell and which is taken up by the double stranded DNA in living germ or embryo cells.

Stains belonging to the class of bis-benzimidazole fluorescent dyes have been found to be particularly useful in this method. This class of compounds has the basic structure: 2073 A series of stains in this class is available from Hoechst AG, Frankfurt, Germany. Compounds in this series include: Hoechst No. R R' 32020 ch3-n< -ci 32021 ch3-n< "och 3 33258 ch3-n< -oh 33342 ch3-n< -oc2h 33662 c2h5-nc "och 3 34580 38312 ch3-n< h-cc -n'ch wnch -ci 38317 h-c< -nh2 Studies by S. A. Latt et al., "Spectral Studies on 33258 Hoechst and Related Bisbenzimidazole Dyes Useful for Fluorescent Detection of Deoxyribonucleic Acid Synthesis," Journal of Histochemistry and Cytochemistry, Vol. 23, pp. 24-33 (1976), demonstrate the similarity of the series of compounds listed above and disclose that these compounds are useful in the detection of DNA synthesis in somatic cells.

The Examples that follow demonstrate that Hoechst stain Nos. 33258 and 33342 are useful in the technique disclosed herein. The chemical name of Hoechst stain No. 33258 is 2 — 12-(4-hydroxyphenyl)-6-benzimidazoyl]-6-(l-methyl-4-piperazyl)-benzimadazole. The chemical name of Hoechst stain No. 33342 is 2-[2-(4-ethoxy phenyl)-6-benzimidazoyl]-6-(l-ethyl-4-piperazyl)-benzimadazole. It is expected that the related bis-benzimidazole compounds will all be useful for the selective visualization of genetic material in living germ or embryo cells. Of the two compounds actually tested, Hoechst stain No. 33342 significantly outperformed Hoechst No. 333258 and therefore is the preferred reagent. It has 5 been noted, however, that the cells of the various species will take up different stains with differing efficiency. For example, Hoechst No. 33258 was taken up rapidly by mouse embryos, but quite slowly by bovine embryos.

Other fluorescent stains meeting the requirements 10 described above, that is, DNA-binding fluorochromes which may be used as vital stains, are expected to be useful with this method as well. Examples include, but are not limited to DAPI (416-diamidino-2-phenylindole), and DIPI (a compound similar to DAPI).

The method by which the genetic material in gametes or embryo cells is stained essentially entails exposure of the cells to a suitable fluorescent stain, incubation and exposure to ultraviolet light. The stain dosage and the incubation period can be varied, depending on the stain 20 used and the purpose for which visualization is desired.

No false positives have been found using this staining technique; that is, the procedure has not been found to significantly stain materials other than nuclear or pronuclear DNA. It has been noted, however, that 25 overstained cells will exhibit some background staining. The technique has been found to result in some false negatives; that is, some cells failed to fluoresce which were found by other methods to contain nuclei or pronuclei. It is not considered that this will hinder or 2073 9 3 compromise the usefulness of the technique in either a laboratory or commercial setting.

Egg cells (oocytes), one-cell fertilized eggs or pre-implantation embryos are obtained from mature female animals by conventional means which will preserve the health of the cells. Sperm cells (spermatozoa) are obtained from the ejaculate of mature male animals or from excised testes, epididymis or part of the male duct system. Germ cells or gametes (oocytes and spermatozoa) are haploid cells which will unite to form a fertilized egg. The fertilized egg (containing the male and female pronuclei) then undergoes syngamy (the union of the male and female pronuclei to produce a zygote nucleus), followed by a series of cell divisions. As the embryo undergoes a series of mitotic divisions, it develops through the morula stage to the blastocyst stage, when tissue differentiation and specialization occur. Ultimately, this leads to formation of the fetus and placenta, which become implanted on the uterine wall.

It is the embryo in the pre-implantation stages, as well as the germ cells and one-cell fertilized eggs, which are the primary focus of this invention. These very early cells are the subjects of intensive research efforts. The gene and nuclear transfer techniques described above utilize fertilized egg cells and early embryo cells. The technique has proven useful in other embryological research as well. For example, it enables researchers to study the development of in vitro fertilized e^ggs, e.g., the symmetry of nuclear and cytoplasmic division, the appearance of a single nucleus in each blastomere, the number of cells in the embryo after each division, etc. 2073 In addition, it is useful for studying the health and motility of spermatozoa.

The germ or embryo cells preferably are placed in culture medium appropriate for maintaining the health and viability of the cells. The preferred culture medium for a given application, e.g., for cells of a particular species and at a particular developmental stage, will be known to one skilled in the art. Several suitable media are given in the Examples. Sperm may be examined in semen, in semen with conventional extenders such as egg yolk or milk extender, or in culture medium.

The stain preferably is used in solution. This allows for more precise regulation of the dosage, as well as allowing very dilute strengths of the stain to be used. It is believed that lower dosages may have less tendency to cause genetic damage to the cell.

A stock solution of the stain may be prepared using any suitable solvent; that is, any solvent for the stain which will not adversely affect the living cells.

Examples include, but are not limited to Dulbecco's Phosphate Buffered Saline (DPBS), TALP, Whittens or Eagles media, or any other media or solvent compatable with the continued viability of the cells. The stain stock solution preferably should be stored at refrigerated temperatures, preferably about 0°C to 4°C, in order to reduce the possibility of contamination. However, it should be noted that freezing generally will not be desirable as it causes the salts to precipitate out of the solution.

The strength of the stain stock solution may be varied, as desired. A stronger solution will stain genetic material in a shorter period, whereas a weaker solution will require a longer incubation period before 2073 9 the material fluoresces. In addition, the stain uptake rate varies with th« species, with bovine and porcine cells requiring a much longer incubation period than murine cells. As noted above* lower dosages of the stain may be preferred to maintain the health and viability of the cells.

For example, using a stock solution of 20 yg stain per ml DPBS, as little as 1 yl in 49 yl of media (50 yl total volume) containing mouse one-cell embryos resulted in distinct fluorescence of pronuclei and at least one polar body within 15 minutes. Using bovine one-cell embryos and 20 yg/ml stain stock solution at a dosage of 2 yl in 48 yl of culture media containing embryos, polar bodies fluoresced within 30 minutes, while the pronuclei were not visible for 1 to 2 hours. As can be seen with reference to the Examples, the dose and incubation period may be varied as desired for various experimental or other purposes.

The stained structures may be visualized microscopically by exposure to ultraviolet light. Light at an excitation wavelength of about 350 nm and emission about 460 nm has been found to be suitable. Nuclei and pronuclei appear as stained blue areas within the cell cytoplasm. Sperm have no discrete pronuclei; the DNA is condensed and packed into the head of the sperm where it may be visualized by this method. Where pronuclei are referred to, it is meant to include the DNA of sperm, unless otherwise specified. Depending on the amount of stain taken up by the DNA, which is a function of dose and time, the intensity will vary from very faintly to brightly fluorescing.

U/ J The following Examples are given for illustrative purposes only and are not meant to limit the invention described herein except as defined by the appended claims. The stock solutions and culture media used in the Examples 5 were prepared as indicated below.

Preparation of Dulbecco's Phosphate Buffered Saline (DPBS) Dulbecco's Phosphate Buffered Saline (DPBS) was prepared as follows: Solution #1 8.00 gm NaCl 0.20 gm KC1 1.15 gm Na2HP04 0.20 gm KHTPO. 800.00 ml deionized distilled water Solution #2 0.1 gm CaClj 100.0 ml deionized distilled water Solution #3 0.1 gm MgCl2 100.0 ml deionized distilled water Each solution was steam autoclaved separately at 120-125°G for 20 minutes and then cooled. The three solutions were mixed in a one liter volumetric flask, and the total volume adjusted to one liter with autoclaved deionized distilled water. The DPBS stock solution was refrigerated at about 4°C.

When DPBS was used to wash cells, 5 mg (0.1 ml) gentamycin sulfate stock solution first was added to 100 ml DPBS. The gentamycin stock solution was 50 mg gentamycinSO^ per ml NaCl.

Preparation of Stain Stock Solutions Stain stock solutions using Hoechst No. 33342 were prepared in the following concentrations: vg/ml DPBS 20 lig/ml DPBS 60 vg/ml DPBS Stain stock solutions using Hoescht No. 33258 was prepared in the following concentrations: yg/ml DPBS 20 Mg/ml DPBS The stair stock solutions were stored at 4°C.

Preparation of TALP (Tyrode-Albumin-Lactate-Pyruvate) Medium Preparation of stock solutions for use in making up TALP medium was as follows: Ingredient Quantity In: NaCl 9 .210 gm 1.0 1 KC1 1 .237 gm 100 ml CaCl2(2H20) 1 .3 32 gm 100 ml MgCl2(6H20) 2 .436 gm 100 ml NaHC03 1 .403 gm + 1 mg 100 ml phenol red Glucose .310 gm 100 ml Na Lactate 1 .0 ml DL-Lactic Acid* NaH2P04(H20) 28 . 0 mg ** H -O H,0 H,0 h;o H*0 2073 9 *DL-Lactic acid, sodium salt as 60% syrup (Sigma Chemical Co.) The 1.0 ml lactic acid syrup was rinsed into the 35 ml H20 until it was in solution. One drop phenol red was added. The pH was adjusted to approximately 7.4 with NaOH (overshoots may be corrected by adding a very small drop of lactate).

**The 28 rag NaH2P04(H20) was mixed with 10 ml of the Glucose stock solution as prepared above.

The ingredients for each stock solution were mixed, Millipore filtered (0.22 u pore size) into sterile bottles, and refrigerated at about 4°C.

The TALP medium was made up as needed by mixing the following quantities of stock solutions: Stock NaCl KC1 CaCl2(2H20) MgCl2(6H20) NaHCOo NaH2P04(H20) Glucose Na Lactate Stock Cone, in Cone.

Medium Quantity 157.0 AM 114.00 mM to 100 ml 166.0 mM 3.16 mM 1.90 ml 120.0 mM 2.00 mM 1.70 ml 120.0 mM 0.50 mM 0.41 ml 167.0 mM .00 mM .00 ml .5 mM 0.35 mM) together 1.70 ml 29 5.0 mM .00 mM) 150.0 mM .00 mM 6.70 ml The indicated quantities were mixed together with sufficient NaCl to bring the final volume to 100 ml and 6.5 mg penicillin (100 i.u./ml) and 1.0 mg phenol red were added. The resulting mixture was Millipore filtered (0.22 y pore size) into a sterile bottle.

On the day of use, the TALP medium was supplemented as follows, depending on the desired use: 2 0739 3 1. Fertilization Medium - 6 mg Bovine Serum Albumin (BSA) (fatty acid free) and 10 yl Na pyruvate stock per ml TALP was added and Millipore filtered. (BSA from Sigma Chemical Co.) or 2. Maturation Medium - to 4.5 ml TALP, 500 yl fetal bovine serum (10%) , 50 yl Na pyruvate stock and 10 mgFSH/ml TALP were added and Millipore filtered.

Preparation of Whitten's Medium Ingredient Quantity NaCl 514.0 mg KC1 36.0 mg KH2 PO4 16.0 mg MgS02 (7H20) 29.0 mg NaHCO 190.0 mg Na Pyruvate 3.5 mg Ca Lactate (5H2O) 53.0 mg Glucose 100.0 mg K Penicillin 8.0 mg StreptomycinS04 . 0 mg The medium was made up by adding the listed ingredients to a 100 ml volumetric flask. Next, 0.37 ml of 60% Na Lactate syrup (Sigma Chemical Co.) and 0.1 ml of 1% phenol red were added. The final volume was adjusted to 100 ml with deionized distilled water. The medium then was Millipore filtered (0.22 y pore size) and stored at 4°C. On the day of use, the medium was supplemented with Bovine Serum Albumin (BSA) (Sigma Chemical Co.) by adding 1.5 mg BSA per ml medium.

EXAMPLE I Mouse embryos were used in this Example to verify the specificity of the stain for genetic material in living 2073 9 one-cell embryos because the pronuclei of this species are visible in the unstained state. The embryos were obtained from mature female mice which had bred the previous night. The animals were sacrificed by cervical dislocation and the ovaries and oviduct excised. The oviduct was dissected free and one-cell embryos recovered by locating them in the oviduct as a bulging area and puncturing the oviduct wall with fine needles and fine scissors. The cumulus cells were removed by adding 100-200 yl hyaluronidase solution (10 mg/mL DPBS) to the area of the dissected oviduct and allowing it to sit for 10 minutes. The one-cell embryos were recovered by pipetting, and were washed three times in a solution of 3 mg Bovine Serum Albumin (BSA)/ml DPBS before adding them to Whittens medium. The BSA was purchased from Sigma Chemical Co.

Individual drops of Whittens medium were placed on a plastic culture dish. The drops were scratched to cause them to adhere to the plate and then were overlaid with oil. The one-cell embryos then were placed in the drops. The pronuclei were visualized using light microscopy before being stained according to the method of this invention.

The 20 yg/ml strength stain stock solution of Hoechst No. 33342 was used for this Example. It was desired to give the one-cell embryos dosages of 0, 1 yl, 2 yl, 5 yl or 10 yl of stain stock solution in a total medium-embryo-stain volume of 50 yl per drop. It therefore was necessary to place the medium-embryo drops on the slides in sizes to accommodate the desired stain 2073 dosage. That is, 49 yl medium-embryo for 1 yl stain dosage, 48 yl medium-embryo for 2 pi stain dosage, etc. The embryos were stained at 37°C, pH 7.2 in 5% C02-in-air. They were left in the stain while being visualized.

The pronuclei and polar bodies of the one-cell mouse embryos were visualized using Nikon epi-fluorescence attachment No. TMD-EF for Inverted Microscope DIAPHOT-TMP (Nikon). This system uses a high pressure mercury lamp. The UV filters used were by Nikon. This system permits 10 visualization with light and fluorescence simultaneously so that it was possible to positively identify the areas of fluorescence as the pronuclei and polar bodies*.

All of the 66 embryos stained in this manner showed distinct fluorescence of both pronuclei and at least one 15 polar body within 15 minutes of initial exposure to the stain, regardless of dosage. Frequently, only one polar body was evident as the second polar body degenerates with time.

Next, 24 one-cell mouse embryos were stained in the 20 same manner as above, using the low dose only (1 yl stain solution with 49 yl medium-embryo drop). Twenty-three of the 24 fluoresced within 5 minutes after staining.

EXAMPLE II The procedures of Example I were repeated using 25 one-cell hamster embryos and a dosage of 10 yl stain solution per 50 yl total drop volume. The first polar body stained after 15 minutes exposure to the stain. The pronuclei did not fluoresce until 30 minutes exposure- Up 2073 9 to 45 minutes exposure was required for complete visualization of the two pronuclei arid at least one polar body in each egg. As in Example I, both polar bodies did not always stain.

EXAMPLE III The procedures of Example I were repeated using matured bovine oocytes, one-cell and 2-cell embryos. Ovaries were obtained from a slaughterhouse. The oocytes were aspirated from the follicles. The cumulus cells were 10 removed prior to staining with hyaiuronidase and pipetting as in Example I.

Some of the recovered oocytes were used for staining, as described below, and some were used for i^j vitro fertilization. The oocytes were incubated for 24 hours at 15 39°C in TALP maturation medium arid then incubated with ejaculated bull semen in TALP fertilization medium. One-cell fertilized egg cells were recovered after the 24 hour incubation, washed in DPBS-gentamycin solution and resuspended in fresh TALP fertilization medium. Two-cell 20 embryos were prepared in the same manner, with recovery after 48 hours incubation with sperm and fertilization medium, and then washed and resuspended in fresh fertilization medium.

Stain stock solution with a concentration of 20' yg 35 Hoechst No. 33342 per ml DPBS was used for this Example. The procedures of Example I were used with stain dosages of 2 yl and 5 yl stain per 50 yl total volume of medium-embryo-stain. The stain-culture mixtures were incubated at 39°C, mounted on glass slides under cover ;0 slips, and visualized under ambient conditions. © 207393 Polar bodies were visible within 30 minutes after the stain was added. The pronuclei of the oocytes and one-cell embryos and the nuclei of the two-cell embryos were visible after one to two hours or more.

The stained cells then were mounted, fixed and cleared to provide a cross-check on the chromatin present. The slides with the stained embryos and oocytes were placed in coplin jars containing approximately 40 ml of acetic acidsethanol (1:3 (vol/vol)) and allowed to clear 10 overnight at room temperature. Chromatin was stained with 1% aceto-orcein (wt/vol) (1% orcein stain in 60:40 water-acetic acid solution). The chromatin was visualized by light microscopy using Nemarsky and phase optics.

It was found that approximately 15% of oocytes and 15 early one-cell embryos contained metaphase plates or pronuclei although they had failed to fluoresce upon exposure to the bis-benzimidazole stain. With the two-cell embryos, no instances were seen in which the nucleus failed to fluoresce. The stain did not give any 20 false positives; all embryos which fluoresced were found to contain chromatin when evaluated by the second method. The chromatin was present in one of the following forms: (1) scattered or metaphase plate for oocytes (including germinal vesicles for the immature oocytes), (2) pronuclei 25 for fertilized egg cells, (3) nuclei for embryo cells, or (4) condensed sperm heads which penetrated the oocyte but did not undergo normal decondensation to form the male pronucleus. o 2073 EXAMPLE IV The basic procedures of Example III were repeated using porcine oocytes, one-cell fertilized eggs and two-cell and four-cell embryos. The oocytes were 5 recovered from bred sows by surgical fLushing of the oviducts and suspended in TALP maturation medium. The 60 g/ml strength stain stock solution of Hoechst No. 33342 was used according to the procedures of Example I with stain dosages of 0, 1 yl, 5 yl and 10 ul per 50 yl total 10 volume of medium-embryo-stain. These mixtures were incubated at 39°C for 3 hours.

The metaphase plates and polar bodies of oocytes which had not been fertilized and fertilized eggs stained very distinctly# with pronuclei appearing less distinct. In 15 addition, sperm heads which had penetrated the zona of oocytes showed fluorescence. The nuclei of two-cell and four-cell embryos showed very distinct fluorescence. It was found that the 10 yl dosage resulted in high levels of background staining.

The stained cells were mounted, fixed and cleared as in Example III, except that they were cleared in acetic acid:ethanol for 48 hours. The results were similar to those in Example III. About 40% of the oocytes and one-cell embryos had failed to fluoresce, although no 25 false positives were observed. The multi-cell embryos were routinely stained with the Hoechst stain. 2073 9 EXAMPLE V The ill vitro development of embryos exposed to Hoechst fluorescent stain No. 33342 was evaluated with mouse embryos at the two-cell and morula stages. The embryos were obtained and prepared by the method described in Example I. They were stained with 10 yl doses of stain solution (20 yg/ml concentration, resulting in a dosage of 0.2 yg per 50 yl total drop volume.

The embryos were exposed to the stain for 15 minutes. Next, the embryos were washed three times in DPBS-gentamycin and cultured in Whitten's medium.

Of 26 two-cell mouse embryos exposed to the stain, 16 or 6 5% developed to the morula stage. This compares with 21 of 26 controls (80%) exposed to no stain, i.e., incubated for 15 minutes with 10 yl doses of DPBS alone.

When morula-stage embryos were exposed to the stain, 23 of 44 (51%) developed to blastocysts. Of the controls (DPBS only), 23 of 31 (74%) developed to blastocysts.

Example VI The basic procedures of Example I were repeated, using Hoechst No. 33258 (10 yg/ml and 20 yg/ml stain stock solutions) to stain one-cell mouse embryos. The dosages were 0, 5 yl, and 10 yl in 50 yl total medium-embryo-stain volume. The embryos were stained under ambient conditions for one-half hour before being examined. The pronuclei and polar bodies fluoresced and were positively identified using light microscopy as in Example I. It was found that the 10 yl dosage of this stain did not routinely cause overstaining or background staining.

Example VII Bovine spermatozoa were stained with Hoechst stain No. 33342 in order to assess sperm motility and tracking patterns. Freshly ejaculated bull semen was washed with DPBS and centrifuged. The resulting pellet was suspended in TALP medium and exposed to 20 jjg/ml concentration Hoechst No. 33342 stain stock solution at a dosage of 3 yl/50 pi. The medium-sperm-stain mixture was incubated for 15 minutes at 39°C and observed.

Following staining, the fluorescing sperm were visualized using the Nikon fluorescence system described in Example I. A camera in the microscope's optical system was used to take color time lapse photographs at exposures of from about 10 to 20 seconds. The tracks produced by the fluorescent DNA on the developed photographs were analyzed as to rate and pattern of sperm motility. The procedure was repeated with the addition of cumulus-oocyte complexes to the medium, and the resulting photograph was analyzed as to the effect of these cells on sperm motility rate and pattern.

Example VIII This example was conducted to observe cleavage in porcine embryos of various stages. Embryos were obtained from the reproductive tracts of superovulated sows 48-120 hours after the onset of estrus. The reproductive tracts were recovered after slaughter and were flushed in the laboratory to remove the embryos. Embryos recovered ranged from one-cell (48 hours) to multicell morula (120 hour) embryos. Following two washings in a DPBS-BSA 2073 9 3 solution, the embryos were either cultured in Whitten's medium (control) or stained and then cultured in Whitten's medium.

The 60 yg/ml strength stain stock solution of Hoechst 5 No. 3334 2 was used for this Example. The embryos were incubated for one hour at 39°C in either 1, 5 or 10 yl stain solution per 50 yl. This was followed by three 30-minute washings in clean DPBS (1.5 mg/BSA/ml). The washed embryos were cultured for 24 hours in Whitten's 10 medium (1.5 mg/BSA/ml) and then were evaluated for development (completing cleavage division). The results are shown in Table 1.

Table 1 Stage Dose Control 1 Ml 5 yl 10 yl 1 or 2 Cell 6/12(50%) — 2/14(14%) 1/12(8.3%) 4 Cell 28/36(77%) 17/27(62%) 14/37(37%) Multicell 15/16(94%) -- 13/23(56%) The principles, preferred embodiments and modes of 20 operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since these are to be regarded as illustrative rather than 25 restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention. 207393

Claims (1)

1. WHAT WE CLAIM IS: 1. A method of preparing living mammalian cells selected from the group consisting of germ cells, one-cell fertilized egqs and pre-implantation embryo cells, for genetic manipulation by staining the DNA of said cells, comprising: 2. The method of CLaim 1 in which said cells or eggs are in culture medium. 3. The method of Claim L in which the fluorescent stain is selected from the group comprising DAPI, DIPI and bis-benzimidazole compounds. 4. The method of Ci nm I in which said stain is used in solution. 5. The method of Claim 3 in which the dosage of said stain is from .001 pg to -10 pg per 50 pi culture containing sai«1 m^inino I ian cells. 6. A mammaLian c«;ll from the group consisting of germ cell:., oik; cell fertilized eggs and pre-implantation embryo eelIs, the cell having its DNA stained in accordance with the method of Claim 1. 7. The cell of Claim 6 in which tne fluorescent dye is selected from the group comprising DAPI, DIPI and bis-benzimidazole compounds. (a) contacting said cells with a fluorescent stainr and (b) incubatinj unti I said DNA fluoresces when exposed to ultraviolet light. -24- 5^ i i 3 1 1 3 5 1 3 1 1 3 1 207393 8. The cell of Claim 7 in which the stain is used in solution. 9. The cell of Claim 7 in which the dosage.of said stain is from .001 pg to 10 pg per 50 pi culture containing said cell. v . j 10. a method of preDarinq pronuclei in livingmammalian pronuclear, cells for genetic manipulation oy staining the UNA of said pronuclei, comprising (a) contacting living mammalian pronuclear cella with a fluorescent dye, and (b) incubating until the pronuclei fluoresce. 11. The method of Claim 10 in which said living mammalian pronuclear cells are bovine or porcine one-cell fertilized eggs. 12. The method of Claim 10 in which said cells are in culture medium. 13. The method of Claim in which the fluorescent dye is selected from the group comprising DAPI, DIPI and bis-benzimidazole compounds. 14. \ iphe method of Claim 13 in which the said dye is in solution. 11 1 |20Ffei?87r -25- W £ I v.V*' 3 L 3 1 1 3 5 7 9 11 1 1 3 / ■207393 15. The method of Claim *3 in which the dye dosage of said stain is from .001 pg to 10 pg per 50 pi culture drop containing said pronuclear cells. 16. a living mammalian pronuclear cell having its pronucleus stained in accordance with the method of Claim 11. 17. a process for removing genetic material from a living mammalian germ cell, one-cell fertilized egg or pre implantation embryo ceLl. comprising: (3) suspending living cells or eggs in a medium, (b) contacting the suspension with a fluorescent dye, (c) incubatmq until the genetic material t Luoresi.v:; upun illumination with ultraviolet light, and (d) removing :;.juJ genetic material from said cells uiiimj micromanipuLation techniques. 18. The process of Claim 17 in which said pronuclear cells are bovine or porcine one-cell fertilized eggs. 19. The process oi 'Jl.um 17 in which the fluorescent dye is selected from tin? <.jiuup comprising DAPI, DIPI and bis-benzimidazole compounds. fa Z20F£BI987 -26- 207393 20. The process of Claim 19 in which the dye is in solution. 21. The process of Claim 19 in which the dye dosage is from .001 |ig to 10 pg per 50 pi culture drop containing said cells or eggs. 22. Genetic material which has been isolated by the process of Claim 17. 23. Bi process for tr.in:?Lerring genetic material into a pronucleus ofc a living ni.immalian pronuclear cell compr isinq: (a) contacting said cell with a fluorescent dye (b) incubating until the DNA of the pronuclei foresee, and (c) transferring the desired genetic material into a pronucleus ^ pronuclear cell by micromanipulation techniques. 24. The process ul CL.utn 23; in which said pronuclear cells are bovine or pui.-ini' one-cell fertilized eggs. 25. The process m CL.jim 23 in which said cells are in culture medium. 26. The process f>l: CLaim 23 in which said dye is selected from the gr«>»i|» comprising DAPI, DEPI and bis-benzimidazo le oc>iii|»<»wh«Im. 27. The process o I CLum 26i in which the dye is in solution. 207393 23. 'The process of Claim 26 in which the dye dosage is from .001 pg to 10 yg per SO pi culture drop containing said cell. , ??•. A genetically altered, living mammalian cell haviny genetic material inserted into it by the process of Claim 23. 30. A genetically ai tered 1 iving cell or clone derived from the cell of claim 29. 31. The procK'fj.s ot L<jitn 23 in which said genetic material is isolated by u process comprising: (a) contacting the cell containing said genetic material with a fluorescent dye, (b) incubating until. said genetic material fluoresces and (c) removing said genetic material from the cell by micromanipulation techniques. 32. The process <»l • .*I •«iiu 31 in which said dye is selected from the group comprising DAPI, DIPI and bis-benzimidazole compounds. 33. The process of Claim 32 in which the dye is in solution. 34. The proc«»ns ol "l.iim 32 in which the dye dosage is from ,00L |ig to 10 yy per 50 yl culture containing the ccLl com iinuiij said genetic material. o 207393 1 35. Fluorescent mammalian DNA in living mammalian cells selected from the group consisting of germ cells, 3 one-cell fertilized eggs and pre-implantation embryo cells, produced by a process comprising: 5 (a) contacting the cells with fluorescent dye and (b) incubating until the DNA fluoresces when 7 exposed to ultraviolet light. 1 36. The fluorescent mammaLian DNA of Claim 35 in which the living mammalian cells used to prepare said 3 fluorescent DNA are bovine or porcine one-cell fertilized eggs and said fluorescent DNA comprises the pronuclei of 5 the cells. 1 37. The fluorescent minima I ian DNA of Claim 35 in which the fluorescent dye is selected from the group 3 comprising DAPI, DIPI and bis-benzimidazole compounds. 1 38. The floucescent mammalian DNA of Claim 37 in which said stain is used in solution. 1 39. The fluorescent niauiiiMi. ian DNA of Claim 37 in which the dosage of ,;ai1 .si.iin is from . OOlpg to 3 10 pg per 50 nL cuiliir<; containing said cells. ' ' |T20 FEB 1987 -3o- 207393 40. A method as claimed in any one of claims 1 to 5 when performed substantially as hereinbefore described with reference to any example thereof. 41. A cell as claimed in any one of claims 6 to .9 substantially as hereinbefore described with reference to any example thereof. 42. A method as claimed in any one of claims 10 : to IS when performed substantially as hereinbefore described with reference to any example thereof. 43. A cell as claimed in claim .16 [ substantially as hereinbefore described with reference to any example thereof. 44. ] A process as claimed in any one of claims 17 to 21 \ substantially as hereinbefore described with reference to any example thereof. 45. A process as claimed in any one of claims 23 to 28 substantially as hereinbefore described with reference to any example thereof. 46. A cell as claimed in any one of claims 29 or 30 </C' 32 substantially as hereinbefore described with reference to any example thereof. 47. A process as claimed in any one of claims 31 to 34 36 substantially as hereinbefore described with reference to any example thereof. 48. A fluorescent mammalian DNA as claimed in any one of claims 35 to 39 1 substantially as hereinbefore described with reference to any example thereof. DATED THIS t^DAY OF A. J. PARK & SON PER agents for the applicants 207393 31 49. A method as claimed in any one of claims 11 to 16 when performed substantially as hereinbefore described with reference to any example thereof. 50. A cell as claimed in claim 17 or 18 substantially as hereinbefore described with reference to any example thereof. 51. A process as claimed in any one of claims 19 to 23 substantially as hereinbefore described with reference to any example thereof. 52. A process as claimed in any one of claims 25 to 30 substantially as hereinbefore described with reference to any example thereof. 53. A cell as claimed in any one of claims 31 or 32 substantially as hereinbefore described with reference to any example thereof. 54. A process as claimed in any one of claims 33 to 36 substantially as hereinbefore described with reference to any example thereof. 55. A fluorescent mammalian DNA as claimed in any one of claims 37 to 41 substantially as hereinbefore described with reference to any example therteof. 56. A method as claimed in any one of claims 42 to 46 substantially as hereinbefore described with reference to any example thereof. DATED THIS DAY OF (V\ct/t2p-\ WS <4" A. dw PARK & SON