Claims
- 1. An isolated nucleic acid molecule comprising
(a) a first class switch region (S1) nucleotide sequence of an upstream immunoglobulin locus under transcriptional control of a first promoter; (b) a second class switch region (S2) nucleotide sequence of an immunoglobulin locus downstream of said upstream Ig locus under transcriptional control of a second promoter, wherein said S2 sequence serves as a region-specific substrate for class switch recombination (CSR); (c) a reporter gene nucleotide sequence encoding a reporter molecule, interposed between said S1 and S2 sequences in reverse transcriptional orientation, and (d) a promoter, downstream of said nucleotide sequence encoding said reporter molecule, allowing the expression of said reporter molecule only following CSR between said S1 and S2 sequences.
- 2. The nucleic acid molecule of claim 1 wherein said S1 is an Sμ sequence and said S2 is an Sγ2 sequence.
- 3. The nucleic acid molecule of claim 1 wherein said S1 is an Sμ sequence and said S2 is an Sε sequence.
- 4. The nucleic acid molecule of claim 2 wherein said S1 and S2 sequences are G-rich switch region DNA sequences.
- 5. The nucleic acid molecule of claim 3 wherein said S1 and S2 sequences are G-rich switch region DNA sequences.
- 6. The nucleic acid molecule of claim 1 wherein said nucleic acid in part (c) and said promoter in part (d) are under control of an internal ribosome entry site (IRES).
- 7. The nucleic acid molecule of claim 1 wherein said nucleic acid in part (c) encodes a Green Fluorescent Protein (GFP) molecule.
- 8. The nucleic acid molecule of claim 1 wherein said nucleic acid in part (c) encodes a reporter molecule selected from the group consisting of β-galactosidase, luciferase, and secreted alkaline phosphatase (SEAP).
- 9. The nucleic acid molecule of claim 1 wherein said first and second promoters are non-inducible constitutive promoters.
- 10. The nucleic acid molecule of claim 9 wherein said first promoter is a CMV promoter.
- 11. The nucleic acid molecule of claim 9 wherein said second promoter is an SV promoter.
- 12. An isolated nucleic acid molecule comprising
(a) a human Sμ nucleotide sequence under control of a CMV promoter; (b) a human Sγ2 nucleotide sequence under control of an SV promoter; (c) an RSV LTR enhancer/promoter and GFP gene under control of an internal ribosome entry site (IRES), interposed between said Sμ and Sγ2 sequences, in reverse transcriptional orientation, (d) a 5′ splicing donor site from human β-globulin gene, 3′ of said Sμ sequence; and (e) a 3′ splicing acceptor site and Cε1 exon, 3′ of said Sγ2 sequence.
- 13. The nucleic acid molecule of claim 12 further comprising a nucleic acid fragment of a cytokine-inducible promoter for Ig germline transcription, 5′ of said CMV promoter.
- 14. The nucleic acid molecule of claim 13 wherein said cytokine-inducible promoter is an IL-4 inducible Iε promoter.
- 15. The nucleic acid molecule of claim 12 selected from the group consisting of XF-1, XF-5a, XF-8, XF-2a, XF-2b, XF-6a and XF-6b.
- 16. A switch vector comprising a nucleic acid molecule of claim 1.
- 17. A switch vector comprising a nucleic acid molecule of 12.
- 18. A recombinant host cell stably transfected with the switch vector of claim 16.
- 19. A recombinant host cell stably transfected with the switch vector of claim 17.
- 20. The host cell of claim 18 which is a mammalian cell.
- 21. The host cell of claim 20, which is a Chinese Hamster Ovary (CHO) cell.
- 22. The host cell of claim 20 which is a primary human B cell.
- 23. A method of monitoring immunoglobulin (Ig) class switch recombination (CSR), comprising
(a) providing a switch vector comprising
(i) a first class switch region (S1) nucleotide sequence of an upstream Ig locus under transcriptional control of a first promoter; (ii) a second class switch region (S2) nucleotide sequence of an Ig locus downstream of said upstream Ig locus under transcriptional control of a second promoter, wherein said S2 sequence serves as a region-specific substrate for CSR; (iii) a reporter gene nucleotide sequence encoding a reporter molecule interposed between said S1 and S2 sequences in reverse transcriptional orientation, and (iv) a promoter, downstream of said nucleotide sequence encoding said reporter molecule, allowing the expression of said reporter molecule only following switch recombination between said S1 and S2 sequences; (b) stably transfecting a mammalian cell with said switch vector; and (c) monitoring the expression of said reporter molecule in said mammalian cell, wherein such expression indicates CSR.
- 24. The method of claim 23 wherein said mammalian cell is a primary B cell or a B cell line.
- 25. The method of claim 24 wherein said B cell line is a human B lymphoma cell line.
- 26. The method of claim 25 wherein said cell line contains a single copy of said switch vector.
- 27. The method of claim 23 wherein said reporter molecule is Green Fluorescent Protein (GFP).
- 28. The method of claim 27 wherein CSR is monitored by fluorescence microscopy.
- 29. The method of claim 28 further comprising the step of quantifying CSR.
- 30. The method of claim 29 wherein said CSR is quantified by flow cytometry.
- 31. The method of claim 29 wherein said first promoter is a CMV promoter.
- 32. The method of claim 29 wherein said second promoter is an SV promoter.
- 33. The method of claim 31 wherein said switch vector further comprises a cytokine-inducible promoter for Ig germline transcription 5′ of said CMV promoter.
- 34. The method of claim 33 wherein said cytokine-inducible promoter is an IL-4 inducible Iε promoter.
- 35. The method of claim 34 further comprising the step of culturing said cells in the presence of IL-4 and/or a stimulator of CD40 activity prior to monitoring CSR.
- 36. The method of claim 35 wherein said stimulator of CD40 activity is an anti-CD40 monoclonal antibody (mAb) or a CD40 ligand.
- 37. The method of claim 35 further comprising the step of exposing said cells to a candidate molecule, and determining the effect of said candidate molecule on GFP expression.
- 38. A method of monitoring immunoglobulin (Ig) class switch recombination (CSR) comprising
(a) providing a switch vector comprising, under transcriptional control of a promoter and in natural transcriptional orientation,
(i) a first class switch region (S1) nucleotide sequence of an upstream Ig locus; (iii) a second class switch region (S2) nucleotide sequence of an Ig locus downstream of said upstream Ig locus; and (iv) a reporter gene nucleotide sequence encoding a reporter molecule, interposed between said S1 and S2 sequences; (b) incubating said switch vector with a cell-free nuclear extract from Ig-producing cells or cells with Ig-producing potential; and (c) detecting deletion of said reporter gene.
- 39. The method of claim 38 wherein said first class switch region sequence (S1) is an Sμ sequence and said second class switch region sequence (S2) is an Sε sequence.
- 40. The method of claim 38 wherein deletion of said reporter gene is detected following transformation of said switch vector into a recombinant host cell.
- 41. The method of claim 40 wherein said recombinant host cell is a prokaryotic cell.
- 42. The method of claim 41 wherein said prokaryotic cell is an E. coli cell.
- 43. The method of claim 40 wherein said reporter gene is a lacZ gene.
- 44. The method of claim 43 wherein deletion of said reporter gene is detected by counting the white colonies obtained after transformation, in the presence of isopropyl-β-D-thiogalactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal).
- 45. The method of claim 38 wherein said Ig-producing cells are B lymphocytes.
- 46. The method of claim 45 wherein said B lymphocytes are of human origin.
- 47. The method of claim 38 wherein said Ig-producing cells are primary B cells stimulated with CD40.
- 48. The method of claim 38 wherein said S1 and S2 comprise G-rich, tandemly repetitive sequences.
Government Interests
[0001] This invention was made with Government support under Grant Nos. AI40551 and AI15251, awarded by the National Institutes of Health. The Government has certain rights in this invention.