What is claimed is:
1. A method comprising:
associating multi-lingual audio recordings with visual objects in a presentation system accessed by the Internet network, wherein a plurality of visual objects such as charts andor slides controlled by a third party in a server can be accessed by any user of said Internet network, said visual objects being associated each with an audio recording in a particular language selected from amongst several predetermined languages, said method including the steps of:
calling said third party server by a user in charge of the audio recording associated with a visual object via the Public Switched Network,
prompting said user on said user’s telephone to enter said user’s user ID; and
transmitting by a keypad of said telephone said user ID including a first part defining a first language to be used for recording said audio recording, whereby displaying of said visual object by said workstation is automatically synchronized with said audio recording.
2. A method according to claim 1, further comprising the step of entering a number assigned to said visual object associated with said audio recording by the user at said telephone after said user has transmitted said user ID.
3. A method according to claim 1, further comprising the step of transmitting a password by the user at said telephone after this one has transmitted said user ID.
4. A method according to claim 3, wherein said user ID includes a second part defining a directory name wherein the file containing said audio recording is stored.
5. A method according to claim 4, further comprising the step of generating by said third party server a first file name including said directory name and a code defining said first language when ID is received by said third party server.
6. A method according to claim 5, wherein said audio recording in said first language is stored in a data base by using said first file name.
7. A method according to claim 6, further comprising the steps of transmitting from said telephone another ID containing a first part defining a second language to be used for recording said audio recording and a second part defining said directory name, and generating by said third party server another file name including said directory name and a code defining said second language.
8. A method according to claim 6, further comprising the step of copying said audio recording corresponding to said first file name from said data base to a directory at said directory name, said step of copying being activated by the user at said telephone.
9. A method according to claim 8, wherein the file containing said audio recording includes a flag which is set when said audio recording is recorded for the first time or changed and which is reset when said file is copied from said data base to said directory.
10. A presentation system accessed by the Internet network wherein a plurality of visual objects such as charts or slides controlled by a third party in a server can be accessed by any user of said Internet network, said visual objects being associated each with an audio recording in any language selected amongst several predetermined languages; said system comprising:
a third party server including processing means for receiving a user ID from a user telephone,
said user ID comprising a first part defining a first language to be used for recording said audio recording whereby displaying of said visual object by said workstation is automatically synchronized with said audio recording.
11. A system according to claim 10, wherein said third party server comprises a table for determining said language to be used for recording said audio recording from said first part in said user ID.
12. A system according to claim 11, wherein said user ID includes a second part defining a directory name, wherein the file containing said audio recording is stored, and further comprising a directory to store said audio recording at a location determined by said directory name.
13. A system according to claim 11, further comprising a data base wherein said file containing said audio recording is stored after being recorded.
14. A system according to claim 12, wherein said file containing said audio recording is copied in said directory after activation of said processing means by the user at said telephone.
15. An apparatus comprising:
means for associating multi-lingual audio recordings with visual objects in a presentation system accessed by the Internet network, wherein a plurality of visual objects such as charts andor slides controlled by a third party in a server can be accessed by any user of said Internet network, said visual objects being associated each with an audio recording in a particular language selected from amongst several predetermined languages, said means for associating including:
means for calling said third party server by a user in charge of the audio recording associated with a visual object via the Public Switched Network,
means for prompting said user on said user’s telephone to enter said user’s user ID; and
means for transmitting by a keypad of said telephone said user ID including a first part defining a first language to be used for recording said audio recording, whereby displaying of said visual object by said workstation is automatically synchronized with said audio recording.
16. An apparatus according to claim 15, further comprising means for entering a number assigned to said visual object associated with said audio recording by the user at said telephone after said user has transmitted said user ID.
17. An apparatus according to claim 15, further comprising means for transmitting a password by the user at said telephone after this one has transmitted said user ID.
18. An apparatus according to claim 17, further comprising means for generating by said third party server a first file name including said directory name and a code defining said first language when ID is received by said third party server.
19. An article of manufacture comprising a computer usable medium having computer readable program code means embodied therein for causing association of multi-lingual audio recordings with visual objects, the computer readable program code means in said article of manufacture comprising computer readable program code means for causing a computer to effect the steps of claim 1.
20. A computer program product comprising a computer usable medium having computer readable program code means embodied therein for causing a presentation system accessed by the Internet network, the computer readable program code means in said computer program product comprising computer readable program code means for causing a computer to effect the functions of claim 10.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
What is claimed is:
1. A method of preparing a spermatozoon suitable for use in ICSI-mediated transgenesis, wherein the method comprises the steps of:
(a) suspending the spermatozoon in a buffered medium, wherein the buffered medium comprises an ion-chelating agent;
(b) treating the spermatozoon to obtain a membrane-disrupted or demembranated spermatozoon; and
(c) incubating the membrane-disrupted or demembranated spermatozoon with an exogenous nucleic acid for a period of time.
2. The method of claim 1, wherein the spermatozoon is a sperm head.
3. The method of claim 1, wherein the buffered medium is a suspension medium.
4. The method of claim 3, wherein the suspension medium is CZB medium.
5. The method of claim 3, wherein the suspension medium is Tris-HCl buffer.
6. The method of claim 1, wherein the ion-chelating agent is a divalent-ion-chelating agent.
7. The method of claim 6, wherein the divalent-ion-chelating agent is a calcium-chelating agent.
8. The method of claim 7, wherein the calcium-chelating agent is EGTA.
9. The method of claim 6, wherein the concentration of the calcium-chelating agent is between 0.1 mM and about 200 mM.
10. The method of claim 9, wherein the concentration of the calcium-chelating agent is between 1 mM and about 100 mM.
11. The method of claim 10, wherein the concentration of the calcium-chelating agent is between 40 mM and about 60 mM.
12. The method of claim 11, wherein the concentration of the calcium-chelating agent is 50 mM, and the calcium-chelating agent is EGTA.
13. The method of claim 1, wherein the pH of the buffered medium is between 7.2 and 8.6.
14. The method of claim 13, wherein the pH of the buffered medium is between 7.4 and 8.2.
15. The method of claim 14, wherein the pH of the buffered medium is 8.2, the concentration of the ion-chelating agent in the buffered medium is 50 mM, and the ion-chelating agent is EGTA.
16. The method of claim 1, wherein the treatment comprises freezing and thawing the spermatozoon to obtain the membrane-disrupted spermatozoon.
17. The method of claim 1, wherein the treatment comprises freeze-drying and rehydrating the spermatozoon to obtain the membrane-disrupted spermatozoon.
18. The method of claim 1, wherein the treatment comprises extracting the spermatozoon with detergent to obtain the demembranated spermatozoon.
19. The method of claim 1, wherein the exogenous nucleic acid comprises more than one transgene.
20. The method of claim 1, wherein the exogenous nucleic acid comprises a cDNA.
21. The method of claim 1, wherein the exogenous nucleic acid comprises genomic DNA.
22. The method of claim 1, wherein the incubating time period with the exogenous nucleic acid is sufficient to form a close association between the exogenous nucleic acid and the membrane-disrupted or demembranated spermatozoon.
23. The method of claim 22, wherein the incubating time period if about 30 seconds to 5 minutes.
24. The method of claim 23, wherein the incubating time period with the exogenous nucleic acid is about 45 seconds to 3 minutes.
25. The method of claim 24, wherein the incubating time period with the exogenous nucleic acid is about 1 minute to 2 minutes.
26. The method of claim 25, wherein the incubating time period with the exogenous nucleic acid is about 1 minute.
27. The membrane-disrupted or demembranated spermatozoon of claim 1, wherein the exogenous nucleic acid is closely associated with the membrane-disrupted or demembranated spermatozoon.
28. The method of claim 1, further comprising the step of incubating the membrane-disrupted or demembranated spermatozoon with a disulfide reducing agent for a period of time, followed by incubation of the membrane-disrupted or demembranated spermatozoon with the exogenous nucleic acid.
29. The method of claim 28, wherein the disulfide reducing agent is contained in a buffered medium.
30. The method of claim 29, wherein the buffered medium is a suspension medium.
31. The method of claim 30, wherein the buffered medium is a buffered saline solution.
32. The method of claim 28, wherein the disulfide reducing agent is one of DTT, TCEP, TCP, TRX or GSH.
33. The method of claim 32, wherein the disulfide reducing agent is DTT.
34. The method of claim 33, wherein the concentration of DTT during incubation with the membrane-disrupted or demembranated spermatozoon is between 0.1 mM and about 50 mM.
35. The method of claim 34, wherein the concentration of DTT during incubation with the membrane-disrupted or demembranated spermatozoon is between 1 mM and about 5 mM.
36. The method of claim 35, wherein the concentration of DTT during incubation with the membrane-disrupted or demembranated spermatozoon is 1 mM.
37. The method of claim 28, where the incubation time period of the membrane-disrupted or demembranated spermatozoon with the disulfide reducing agent is sufficient to result in partial or complete nuclear decondensation of the membrane-disrupted or demembranated spermatozoon.
38. The method of claim 37, wherein the incubation time period is about 5 minutes to 1 hour.
39. The method of claim 38, where the incubation time period is about 10 minutes to 50 minutes.
40. The method of claim 39, where the incubation time period is about 20 minutes to 40 minutes.
41. The method of claim 40, where the incubation time period is about 30 minutes, and the disulfide reducing agent is DTT in 1 mM concentration.
42. The membrane-disrupted or demembranated spermatozoon of claim 28, wherein the exogenous nucleic acid is closely associated with the membrane-disrupted or demembranated spermatozoon.
43. A method for obtaining a transgenic embryo, comprising the steps of:
(a) coinserting the spermatozoon of claim 1 and the exogenous nucleic acid into an unfertilized oocyte to form a transgenic fertilized oocyte; and
(b) allowing the transgenic fertilized oocyte to develop into a transgenic embryo.
44. The method of claim 43, further comprising the step of transplanting the transgenic embryo into a surrogate mother and allowing the transgenic embryo to develop into a live offspring.
45. A method for obtaining a transgenic embryo, comprising the steps of:
(a) coinserting the spermatozoon of claim 28 and the exogenous nucleic acid into an unfertilized oocyte to form a transgenic fertilized oocyte; and
(b) allowing the transgenic fertilized oocyte to develop into a transgenic embryo.
46. The method of claim 45, further comprising the step of transplanting the transgenic embryo into a surrogate mother and allowing the transgenic embryo to develop into a live offspring.