Dividing of reporter proteins by DNA sequences and its application in site specific recombination
Methods and constructs for inserting an intron into a reporter protein coding sequence in a eukaryotic cell and their application of monitoring and reporting genomic modifications are provided. Various related compositions, cells and kits are also provided.
1. A recombinant nucleic acid construct comprising in order from upstream to downstream:
a promoter sequence;
a nucleic acid sequence encoding a first portion of a reporter protein including an N-terminus, wherein a protein product of the reporter protein is insufficient to provide reporter expression;
a splice donor site;
a heterologous nucleic acid sequence;
a splice acceptor site;
a nucleic acid sequence encoding a second portion of a reporter protein including a C-terminus; and
a poly(A) signal sequence.
2. The nucleic acid construct of claim 1 , wherein the promoter is a nucleic acid sequence capable of driving gene expression of downstream sequences in eukaryotic cells.
3. The nucleic acid construct of claim 2 , wherein the promoter is a polymerase II promoter.
4. The nucleic acid construct of claim 2 , wherein the promoter is selected from the group consists of ubiquitous promoter, cell specific promoter, inducible promoter, and constitutive promoter in eukaryotic cells.
5. The nucleic acid construct of claim 4 , wherein the promoter is selected from the group consists of the CAG (SEQ ID NO: 1), CAGGS, CMV, hCMV, EF1, PGK, FABP, Lck, CamKII, CD19, Keratin, Albumin, aP2, Insulin, MCK, MyHC, WAP, Col2A, Mx, tet, and Trex promoter.
6. The nucleic acid construct of claim 1 , wherein the reporter protein comprises a fluorescent protein, wherein said fluorescent protein is a protein capable of absorption of a higher energy photon and emission of a lower energy photon in eukaryotic cells.
7. The nucleic acid construct of claim 6 , wherein fluorescent protein is selected from the group consisting of blue/UV fluorescent proteins, cyan fluorescent proteins, green fluorescent proteins, yellow fluorescent proteins, orange fluorescent proteins, red fluorescent proteins, far-red fluorescent proteins, Near-IR fluorescent proteins, Long strokes shift fluorescent proteins, Photoactivable fluorescent proteins, Photoconvertible fluorescent proteins, and Photoswitchable fluorescent proteins.
8. The nucleic acid construct of claim 7 , wherein fluorescent protein is selected from GFP, EGFP, and DsRed.
9. The nucleic acid construct of claim 1 , wherein the splice donor site is a DNA sequence at beginning of an intron which can be spliced by splicesome.
10. The nucleic acid construct of claim 9 , wherein the first nucleotide of a 5′ end of the intron is a G.
11. The nucleic acid construct of claim 1 , wherein the splice acceptor site is a DNA sequence at the end of an intron which can be spliced by splicesome.
12. The nucleic acid construct of claim 11 , wherein the last nucleotide of a 3′ end of the intron is a G.
13. The nucleic acid construct of claim 1 , wherein the reporter protein is selected from the group consisting of beta-galactosidase, luciferase, and chloramphenicol acetyltransferase.
14. A nucleic acid construct comprising: a DNA targeting vector comprising, in order, a 5′ homology arm; the nucleic acid construct according to claim 1 , wherein the heterologous sequence comprises a sequence flanked by two recombinant sites; and a 3′ homology arm, wherein the DNA targeting vector further comprises an antibiotic selectable marker gene inserted between the 5′ homology arm and 3′ homology arm.
15. The nucleic acid construct of claim 14 , wherein both of the recombination sites are identical.
16. The nucleic acid construct of claim 14 , wherein both of the recombination sites are not identical.
17. The nucleic acid construct of claim 14 , wherein one of the recombination sites is a mutant recombination site.
18. The nucleic acid construct of claim 14 , wherein at least one recombination site is a wildtype recombination site consists of loxp, frt, rox, Vlox, Slox, attR, attL, attP, attB, or IR/DR sequences.
19. The nucleic acid construct of claim 17 , wherein one of the recombination site is selected from the group consisting of lox511, lox5171, lox2272, M2, M7, M11, lox71, lox66, loxN, loxp 5171, F3, F5, F7, FL-IL10A, Vlox2272, Slox2272, VloxM1, SloxM2, VloxM2, SloxM2, Vlox43R, Vlox43L, Slox1R, or Slox1L.