Prenatal screening

Abstract

The present invention concerns products and methods particularly useful for activating and analyzing non-dividing cell nuclei. The featured products include activating egg extracts, cytostatic factor (CSF) extracts, kits containing these extracts, and a microchamber microscope slide useful in analyzing nucleus activation.

1 . A method for activating a nucleus from a human fetal cell comprising the steps of: 
 a) separating said nucleus from its surrounding cytoskeleton to form a pretreated nucleus, and    b) contacting said pretreated nucleus with an activating egg extract to activate said pretreated nucleus.    
     
     
         2 . The method of  claim 1 , wherein said fetal cell is selected from the group consisting of keratinocyte, trophoblast, erythrocyte and leukocyte.  
     
     
         3 . The method of  claim 2 , wherein said leukocyte is selected from the group consisting of neutrophil, basophile, eosinophil, and granulocyte.  
     
     
         4 . The method of  claim 1 , wherein said separating is carried out using a protease and a non-ionic detergent.  
     
     
         5 . The method of  claim 4 , wherein said protease is trypsin and said detergent is lysolecithin.  
     
     
         6 . The method of  claim 1 , further comprising contacting said pretreated nucleus with CSF extract.  
     
     
         7 . The method of  claim 6 , wherein said nucleus is activated under conditions were the nucleus and its chromosomes do not divide.  
     
     
         8 . The method of  claim 7 , wherein said conditions comprise adding nocodazole to said activating egg extract or said CSF extract.  
     
     
         9 . The method of  claim 8 , wherein said nocodazole is in amount less than 5 μg/ml.  
     
     
         10 . The method of  claim 1 , wherein said nucleus is activated under conditions not suitable for nucleic acid: synthesis.  
     
     
         11 . A method of causing a non-dividing human nucleus to activate, using activating egg extract and CSF extract prepared from hardened eggs comprising: 
 a) incubating said non-dividing human nucleus with said CSF extract prepared from hardened eggs to form a pretreated nucleus, and    b) contacting said pretreated nucleus with said activating egg extract wherein said activating egg extract is prepared from synchronously activated hardened eggs.    
     
     
         12 . The method of  claim 11 , wherein said CSF extract is frozen and thawed before use.  
     
     
         13 . The method of  claim 11 , wherein said activating egg extract is frozen and thawed before use.  
     
     
         14 . The method of  claim 11 , wherein said incubating is performed using a warm-then-cold regime comprising incubating at about 25° C. for at least 30 minutes followed by incubation at about 4° C. for at least 30 minutes.  
     
     
         15 . The method of  claim 11 , wherein said incubating is performed using a warm regime at about 25° C. for at least 30 minutes.  
     
     
         16 . The method of  claim 11 , wherein Ca 2+  is provided in said incubating step.  
     
     
         17 . The method of  claim 16 , wherein said Ca 2+  is provided in an amount greater than 100 μM.  
     
     
         18 . A method for preparing an activating egg extract, comprising the steps of hardening a plurality of eggs, simultaneously inducing said eggs; and preparing an activating egg extract from said eggs wherein said eggs are induced for a length of time such that they have at least 70% of maximal activation DNA synthesis activity.  
     
     
         19 . The method of  claim 18 , wherein said activating egg extract is prepared from a eukaryotic cell.  
     
     
         20 . The method of  claim 19  wherein said eukaryotic cell is an amphibian, yeast, human, echinoderm, mollusc, fish, or chicken cell.  
     
     
         21 . The method of  claim 20 , wherein said eukaryotic cell is a Xenopus cell.  
     
     
         22 . The method of  claim 21 , wherein said eggs are obtained from Xenopus and said length of time is greater than 10 minutes.  
     
     
         23 . The method of  claim 22 , wherein said length of time is between 25 and 30 minutes.  
     
     
         24 . A method of causing a non-dividing cell nucleus to swell comprising the steps of: 
 a) separating said nucleus from its surrounding cytoskeleton to form a pretreated nucleus; and    b) contacting said pretreated nucleus with a CSF extract supplemented with an aqueous solution or a protein kinase inhibitor.    
     
     
         25 . The method of  claim 24 , wherein said nucleus is a human nucleus.  
     
     
         26 . The method of  claim 25 , wherein said CSF extract is a partially purified CSF extract supplemented with an aqueous solution and a protein kinase inhibitor.  
     
     
         27 . The method of  claim 26 , wherein said aqueous solution is an appropriate buffer.  
     
     
         28 . The method of  claim 27 , wherein said protein kinase inhibitor is either 6-dimethylaminopurine or staurosporine.  
     
     
         29 . The method of  claim 28 , wherein said appropriate buffer is provided in an amount to dilute said CSF extract by 25% to 75%.  
     
     
         30 . The method of  claim 27 , further comprising the step of adding an agent to further swell or decondense said nuclei after said step (b).  
     
     
         31 . A method of causing chromosome formation in a non-dividing cell nucleus comprising the steps of: 
 a) separating said nucleus from its surrounding cytoskeleton to form a pretreated nucleus; and    b) contacting said pretreated nucleus with a CSF extract supplemented with a cyclin.    
     
     
         32 . The method of  claim 31 , wherein said cyclin is cyclin- 90.  
     
     
         33 . A method for activating or studying a mammalian sperm cell nucleus comprising the steps of: 
 a) pretreating said sperm cell nucleus to form a pretreated sperm nucleus wherein said pretreating comprises (i) separating said nucleus from its surrounding cytoskeleton by permeabilizing said cell nuclear membrane and incubating in the presence of a protease and (ii) incubating in the presence of a thiol reducing agent; and    b) activating said pretreated sperm cell.    
     
     
         34 . The method of  claim 33  wherein said sperm cell nucleus is a human sperm cell nucleus.  
     
     
         35 . The method of  claim 34 , wherein said activating is carried out by contact with a CSF extract, wherein said CSF extract is supplemented to induce nuclear swelling.  
     
     
         36 . The method of  claim 34 , wherein said activating is carried out by contact with a CSF extract, wherein said CSF extract is supplemented to induce chromosome formation.  
     
     
         37 . The method of  claim 34 , wherein said activating is carried out using an activating extract.  
     
     
         38 . The method of  claim 33 , wherein said sperm cell nucleus is from a transgenic animal.  
     
     
         39 . The method of clam  34 , wherein said human sperm cell nucleus is obtained from either a person infected with a virus or a person not infected with said virus.  
     
     
         40 . The method of  claim 39  wherein, said virus is HIV.  
     
     
         41 . The method of  claim 34 , further comprises the step of analyzing said nucleus by in situ hybridization.  
     
     
         42 . The method of  claim 34 , wherein said step (a) (i) is carried out using a detergent and trypsin.  
     
     
         43 . The method of  claim 34 , wherein said pretreating step further comprises a thiol blocking agent.  
     
     
         44 . The method of  claim 34 , wherein a CSF extract further pretreatment step is carried out between said step (a) and said step (b), comprising incubating the pretreated nuclei in CSF extract.  
     
     
         45 . An activation assay for studying male fertility comprising: 
 a) pretreating a sperm nucleus to separate cytoskeletal protein from nucleic acid,    b) activating said sperm nucleus,    c) measuring activation of said nucleus activated in step (b).    
     
     
         46 . The activation assay of  claim 45 , wherein said pretreating comprises using a detergent, a protease, and a thiol reducing agent.  
     
     
         47 . The activation assay of  claim 46 , wherein said sperm nucleus is a human sperm nucleus.  
     
     
         48 . The activation assay of  claim 47 , wherein said activating is carried out using a CSF extract supplemented with an aqueous solution, a protein kinase inhibitor, or a cyclin.  
     
     
         49 . The method of  claim 47 , wherein said activating is carried out using an activating egg extract.  
     
     
         50 . The method of  claim 49 , further comprising a CSF further pretreatment prior to said activating.  
     
     
         51 . A viral integration assay comprising the steps of: 
 a) pretreating a cell nucleus to separate the nucleus from its surrounding cytoskeleton to form a pretreated nucleus,    b) activating said nucleus and incubating with a viral integration complex containing viral nucleic acid, wherein said integration complex is added before or after said incubating; and    c) measuring integration of viral nucleic acid into nucleic acid of said cell nucleus.    
     
     
         52 . The assay of  claim 51 , wherein said viral nucleic acid is from HIV.  
     
     
         53 . The assay of  claim 52 , further comprising a step, between said step b) and said step c), of adding an agent which inhibits integration of said HIV.  
     
     
         54 . A viral integration assay comprising the steps of: 
 a) constructing a pseudonucleus from a defined DNA template,    b) activating said nucleus and incubating with a viral integration complex containing viral nucleic acid, wherein said integration complex is added before or after said incubating; and    c) measuring integration of viral nucleic acid into nucleic acid of said cell nucleus.    
     
     
         55 . A product for preparing a non-dividing nucleus to activate upon subsequent treatment with activating egg extract, comprising a CSF extract prepared using an eukaryotic cell, wherein said CSF extract is supplemented with Ca 2+ .  
     
     
         56 . The product according to  claim 55 , wherein said Ca 2+  is provided in an amount greater than 100 μM.  
     
     
         57 . A product for causing nuclear swelling comprising CSF extract supplemented with a protein kinase inhibitor and/or an aqueous solution.  
     
     
         58 . The product of  claim 57 , wherein said CSF extract is a partially purified extract.  
     
     
         59 . The product of  claim 58 , wherein said aqueous solution is an appropriate buffer.  
     
     
         60 . The product according to  claim 59 , wherein said supplement is said protein kinase inhibitor and said appropriate buffer.  
     
     
         61 . The product according to  claim 60 , wherein said protease inhibitor is either 6-dimethylaminopurine or staurosporine.  
     
     
         62 . A product for causing chromosome formation in a cell nucleus comprising a CSF extract supplemented with a cyclin.  
     
     
         63 . The product according to  claim 62 , wherein said cyclin is cyclin- 90.  
     
     
         64 . A product for activating a non-dividing nucleus comprising an activating egg extract having at least 70% optimal activation activity.  
     
     
         65 . The product of  claim 64 , wherein said activating egg extract is prepared from one or more eukaryotic egg.  
     
     
         66 . The product of  claim 64 , wherein said activating egg extract is prepared from a plurality of Xenopus eggs synchronously induced for more than 10 minutes.  
     
     
         67 . The product of  claim 66 , wherein said Xenopus eggs are synchronously induced for 25 to 30 minutes.  
     
     
         68 . The product of  claim 66 , wherein said activating egg extract is supplemented with cAMP.  
     
     
         69 . The product of  claim 66 , wherein said activating egg extract is supplemented with a phosphodiesterase inhibitor.  
     
     
         70 . A kit for activating a non-dividing nucleus comprising a first product comprising frozen activating egg extract having at least 70% optimal activation activity and a second product comprising a frozen CSF extract.  
     
     
         71 . The kit according to  claim 70 , wherein said second product is supplemented with Ca 2+ .  
     
     
         72 . The kit according to  claim 70 , wherein said first product and said second product are prepared from hardened eggs.  
     
     
         73 . The kit according to  claim 70 , further comprising a microchamber microscope slide.  
     
     
         74 . A microscope slide comprising; 
 a) an upper surface,    b) a water repellent-means having a defined thickness located upon said upper surface to define a microchamber connected by a channel to at least one well on said upper surface, and    c) said microchamber shaped to enhance flushing of said microchamber.    
     
     
         75 . The microscope slide of  claim 74 , wherein said microchamber is in a teardrop-shape or a pear-shape.  
     
     
         76 . The microscope slide of  claim 74 , wherein two wells are provided at opposite ends of said microchamber and each of said wells are connected to said microchamber by a channel.  
     
     
         77 . The microscope slide of  claim 74 , wherein said microchamber has a defined volume between 5 and 50 μl.  
     
     
         78 . The microscope slide of  claim 74 , wherein said microchamber has defined volume between 10 and 20 μl.  
     
     
         79 . The microscope slide of  claim 74 , wherein said water repellent means is a tape or a coating on said upper surface of said slide.  
     
     
         80 . The microscope slide of  claim 74 , wherein said water repellent means is a TEFLON® coating.  
     
     
         81 . The microscope slide of  claim 74 , wherein said water repellent means is a plastic containing tape.  
     
     
         82 . The microscope slide of  claim 74 , wherein said upper surface is treated to enhance cell growth compared to an untreated slide.  
     
     
         83 . The microscope slide of  claim 74 , wherein said microscope slide is sterile.  
     
     
         84 . The microscope slide of  claim 74 , wherein said microchamber or said at least one well contains an antibody to a human fetal cell.  
     
     
         85 . A method for analysis of growth or manipulation of a cell or cell component comprising providing a microscope slide of  claim 74  and placing said cell or said cell component in said microchamber.  
     
     
         86 . The method of  claim 74 , wherein a fluid is introduced into one said well and is allowed to enter said microchamber and then is removed from said microchamber.