Method of promoting in vitro homologous recombination transfection in mammalian cells using the RecA protein

• US 5,468,629 A
• Filed: 04/13/1993
• Issued: 11/21/1995
• Est. Priority Date: 04/13/1993
• Status: Expired due to Fees

• Current Assignee: Cornelia Calhoun
• Original Assignee: Cornelia Calhoun
• Inventors: Calhoun, Cornelia
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): CARTER, PHILIP
• Application Number:
  US08/047,154
• Time in Patent Office: 952 Days
• Field of Search: 435/4, 435/6, 435/172.1, 435/172.3, 435/240.2
• US Class Current: 435/463
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12Q 1/6841 In situ hybridisation

Abstract

A rapid method for transfection of a cell under physiological conditions suitable to the survival and growth of the cell is disclosed. According to the method, a stable nucleoprotein complex is provided. The nucleoprotein complex comprises a single-stranded DNA sequence in stable combination with RecA protein molecules. Cells to be transformed are cultured in a physiologically suitable medium to which the nucleoprotein complex has been added. As the cells grow and undergo mitosis, the nucleoprotein complex is taken up within some of the cells and becomes integrated into the genome. The method accomplishes transfection without resort to infectious vectors or permeabilization or other manipulation of the cell membrane. According to another object of the invention, a diagnostics method is provided. A directly detectable reporter label or an indirectly detectable ligand is bound to the nucleoprotein complex to provide a DNA probe which then is taken up into the cell and integrates into the cell'"'"'s genome. Upon appropriate treatment the detectable reporter label can be observed or the ligand can be reacted with a suitable detectable reporter molecule to allow visualization and thereby confirm whether the compliment of the DNA sequence in the probe is substantially present in the genome of the cell.

Claims

1. An in vitro method for the transfection of living mammalian cells comprising the steps of:

growing living mammalian cells under physiological conditions suitable for the growth of said cells;

preparing at least one stable nucleoprotein complex with a single-strand DNA sequence of a length of between 200 and 700 bases, and with RecA protein molecules bound to said single-strand DNA sequence, wherein said sequence is substantially identical or complementary to a genomic sequence in said mammalian cells;

mixing said nucleoprotein complex with said living cells, to form an incubation mixture, and;

incubating said incubation mixture for a sufficient period of time to allow said DNA sequence to be transformed into the genome of said cells, said transfection occurring without the additional assistance of a viral vector, calcium phosphate, DEAE-dextran precipitation, lipofection, electroporation, microinjection, or any artificial means for permeating the cell membranes of said cells.

2. The method for the transfection of living mammalian cells according to claim 1, further comprising the step of:

adding to said nucleoprotein complex the components of magnesium acetate of a concentration from between 8 to 20 mM and ATPγ

S, and incubating said incubation mixture for at least 30 minutes.

3. The method for the transfection of living mammalian cells according to claim 2, further comprising the steps of:

incubating said incubation mixture at a constant temperature; and

selecting as said mammalian cells, cells from carcinoma tissue.

4. The method for the transfection of living mammalian cells according to claim 3, further comprising the steps of:

adding to said nucleoprotein complex the following components, in the following approximate quantities, to make a 20.5 μ

l mixture;

6.59 mM magnesium acetate, 24.39 mM sodium acetate, 0.49 mM DTT, 0.24 mM ATPγ

S, and 8.39 μ

M RecA.