Transfection method and uses related thereto

• US 6,951,757 B2
• Filed: 03/04/2003
• Issued: 10/04/2005
• Est. Priority Date: 09/17/1999
• Status: Expired due to Term

• Current Assignee: Whitehead Institute for Biomedical Research
• Original Assignee: Whitehead Institute for Biomedical Research
• Inventors: Sabatini, David M.
• Primary Examiner(s): McKelvey, Terry
• Assistant Examiner(s): VOGEL, NANCY TREPTOW
• Application Number:
  US10/379,130
• Publication Number:
  US 20030228694A1
• Time in Patent Office: 945 Days
• Field of Search: 435/455, 435/320.1, 435/174, 435/458
• US Class Current: 435/455
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 15/88 using microencapsulation, e.g. using amphiphile liposome vesicle
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention features a method of introducing nucleic acid molecules into eukaryotic cells by (a) depositing a nucleic acid molecule-containing mixture onto a surface, (b) affixing the nucleic acid molecule-containing mixture to the surface, and (c) plating eukaryotic cells onto the surface under appropriate conditions for entry of the nucleic acid molecules into the cells.

Claims

1. A method of introducing nucleic acid molecules into eukaryotic cells, said method comprising the steps of:

(a) depositing a nucleic acid molecule-containing mixture onto a surface, wherein said nucleic acid molecule-containing mixture comprises (i) nucleic acid molecules to be introduced into eukaryotic cells and (ii) a gelatin;

(b) affixing said nucleic acid molecule-containing mixture to said surface; and

(c) plating eukaryotic cells onto said surface at a density of 0.3×

10⁵/cm² to 3.0×

10⁵/cm² under appropriate conditions for entry of said nucleic acid molecules into said cells.

2. The method of claim 1, further comprising the steps after step (b) of:

(i) covering said surface bearing said nucleic acid molecule-containing mixture with an appropnate amount of a transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between said nucleic acid molecules and said transfection reagent; and

(ii) removing the non-complexed transfection reagent.

3. The method of claim 1, wherein said nucleic acid molecule-containing mixture further comprises a sugar, a buffer that facilitates nucleic acid molecule condensation, and a transfection reagent.

4. The method of claim 1, wherein each nucleic acid molecule to be introduced is contained in a vector.

5. The method of claim 4, wherein said vector is a plasmid or a viral-based vector.

6. The method of claim 1, wherein said nucleic acid molecules are expressed in said cells.

7. The method of claim 1, wherein said cells are plated at a density of 0.5×

10⁵/cm² to 2.0×

10⁵/cm².

8. The method of claim 1, wherein said nucleic acid molecules are DNA or RNA molecules.

9. The method of claim 8, wherein said nucleic acid molecules are DNA molecules.

10. The method of claim 9, wherein the concentration of said DNA molecules is 0.01 μ

g/μ

l to 0.5 μ

g/μ

l.

11. A method of introducing nucleic acid molecules into eukaryotic cells, said method comprising the steps of:

(a) depositing a nucleic acid molecule-containing mixture onto a surface, wherein the nucleic acid molecule-containing mixture comprises (i) nucleic acid molecules to be introduced into eukaryotic cells and (ii) a gelatin, wherein the concentration of nucleic acid molecules in said nucleic acid molecule-containing mixture is 0.01 μ

g/μ

L to 0.5 μ

g/μ

L;

(b) affixing said nucleic acid molecule-containing mixture to said surface; and

(c) plating eukaryotic cells onto said surface in sufficient density and under appropriate conditions for entry of said nucleic acid molecules into said cells.

12. The method of claim 11, further comprising the steps after step (b) of:

(i) covering said surface bearing said nucleic acid molecule-containing mixture with an appropriate amount of a transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between said nucleic acid molecules and said transfection reagent; and

(ii) removing the non-complexed transfection reagent.

13. The method of claim 11, wherein said nucleic acid molecule-containing mixture further comprises a sugar, a buffer that facilitates nucleic acid molecule condensation, and a transfection reagent.

14. The method of claim 11, wherein each nucleic acid molecule to be introduced is contained in a vector.

15. The method of claim 14, wherein said vector is a plasmid or a viral-based vector.

16. The method of claim 11, wherein said eukaryotic cells are mammalian cells.

17. The method of claim 11, wherein said gelatin is present in said nucleic acid molecule-containing mixture at a concentration ranging from about 0.05% to about 0.5%.

18. A method of introducing nucleic acid molecules into eukaryotic cells, said method comprising the steps of:

(a) depositing a nucleic acid molecule-containing mixture onto a surface, wherein the nucleic acid molecule-containing mixture comprises (i) nucleic acid molecules to be introduced into eukaryotic cells and (ii) a gelatin, wherein said gelatin is present at a concentration from about 0.05% to about 0.5%;

(b) affixing said nucleic acid molecule-containing mixture to said surface; and

(c) plating eukaryotic cells onto said surface in sufficient density and under appropriate conditions for entry of said nucleic acid molecules into said eukaryotic cells.

19. The method of claim 18, further comprising the steps after step (b) of:

(i) covering said surface bearing said nucleic acid molecule-containing mixture with an appropriate amount of a transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between said nucleic acid molecules and said transfection reagent; and

(ii) removing the non-complexed transfection reagent.

20. The method of claim 18, wherein said nucleic acid molecule-containing mixture further comprise a sugar, a buffer that facilitates nucleic acid molecule condensation, and a transfection reagent.

21. The method of claim 18, wherein each nucleic acid molecule to be introduced is contained in a vector.

22. The method of claim 21, wherein said vector is a plasmid or a viral-based vector.

23. The method of claim 18, wherein said eukaryotic cells are mammalian cells.

24. The method of claim 18, wherein said nucleic acid molecules are DNA or RNA molecules.

25. The method of claim 24, wherein said nucleic acid molecules are DNA molecules.

26. The method of claim 18, wherein the concentration of gelatin is from about 0.1% to about 0.2%.

27. A method of introducing nucleic acid molecules into eukaryotic cells, said method comprising the steps of:

(a) depositing a nucleic acid molecule-containing mixture onto a surface, wherein said nucleic acid molecule-containing mixture comprises;

(i) nucleic acid molecules to be introduced into eukaryotic cells,

(ii) a gelatin, wherein said gelatin is present at a concentration from about 0.01% to about 0.05%.

(iii) a sugar,

(iv) a buffer that facilitates nucleic acid molecule condensation, and

(v) a transfection reagent;

(b) affixing said nucleic acid molecule-containing mixture to said surface; and

(c) plating eukaryotic cells onto said surface in sufficient density and under appropriate conditions for entry of said nucleic acid molecules in said nucleic acid molecule-containing mixture into said eukaryotic cells.

28. The method of claim 27, further comprising the steps after step (b) of:

(i) covering said surface bearing said nucleic acid molecule-containing mixtures with an appropriate amount of a transfection reagent and maintaining the resulting product under conditions appropriate for complex formation between said nucleic acid molecules in said nucleic acid molecule-containing mixture and said transfection reagent; and

(ii) removing the non-complexed transfection reagent.

29. The method of claim 27, wherein each nucleic acid molecule to be introduced is contained in a vector.

30. The method of claim 29, wherein said vector is a plasmid or a viral-based vector.

31. The method of claim 30, wherein said eukaryotic cells are mammalian cells.

32. The method of claim 27, wherein said cells are plated at a density of 0.3×

10⁵/cm² to 3.0×

10⁵/cm².

33. The method of claim 27, wherein said nucleic acid molecules DNA or RNA molecules.

34. The method of claim 33, wherein said nucleic acid molecules are DNA molecules.

35. The method of claim 34, wherein the concentration of said DNA molecules is 0.01 μ

g/μ

l to 0.5 μ

g/μ

l.

36. The method of claim 27, wherein said sugar is sucrose ranging in concentration from about 0.1M to about 0.4M.