CZ295047B6 - General-purposes cell system for testing ingression of substances into cell nuclei - Google Patents

Abstract

The present invention relates to a process for preparing a cell testing system for testing ingression of fluorescently labeled synthetic molecules of various chemical natures into eukaryotic cell nuclei. The invented system employs fusion reporter protein ubiquitin, EGFP (Enhanced Green Fluorescent Protein) and amplifiable NLS (Nuclear Localization Sequence), ensuring green fluorescence of the cell nuclei. There is also described a fusion protein sequence, preparation process of a vector encoding such sequence, preparation process of a stable eukaryotic cell line expriming a cloned fusion protein.

Description

Technical field

This solution belongs to the field of molecular genetics and gene therapy, cloning of recombinant DNA molecules, creation of genetically modified, stably transformed cell lines. The invention is closely related to the use of a DNA expression vector carrying a reporter fusion protein that allows the transport of substances across the nuclear membrane to be monitored.

BACKGROUND OF THE INVENTION

GFP (Green fluorescent protein) is a frequently used marker in molecular biology to monitor cell pathways, to study gene expression and protein localization. It was first isolated from a marine animal, the jellyfish Aequorea victoria. The entire gene consists of 238 amino acids. It is able to form fusion proteins at both the C- and N- terminus while maintaining fluorescence.

GFP fluoresces due to the chromophore that results from autocatalytic cyclization of three amino acids in the presence of molecular oxygen. It requires no further substrates or cofactors for its fluorescence (1). The process of intramolecular biosynthesis of the GFP chromophore requires O 2 oxidation, and a byproduct of this reaction is the release of H 2 O 2 molecules in a stoichiometric ratio of 1: 1 based on the number of GFP molecules formed with active chloroform (2). 1. Generated oxygen radicals become toxic to cells with subsequent induction of apoptosis (3).

Several types of GFP mutants are currently known to be grouped according to the type of their chromophore that is responsible for fluorescence. EGFP (ethanced GFP) is a mutant variant of the wild-type GFP in which two amino acids have been substituted, namely the replacement of Phe to Leu at position 64 and Ser to Thr at position 65. EGFP, unlike the wild type other spectral properties, clearer fluorescence, and differ significantly in fluorescence at 37 ° C. While wild type fluoresces at room and lower temperatures, EGFP has the ability to fluoresce at 37 ° C. Otherwise, the mutants are structurally very similar (4).

The use of GFP or EGFP is the subject of many patent solutions. An example is US2002009712. An example of the use of a GFP fusion protein, e.g., a sGas protein, is WO003008435.

Nuclear localization signal (NLS) is one of a number of localization signals that direct nuclear transport of substances. In addition to mitosis, when the nuclear membrane is disintegrated, the transport of substances is controlled by the nuclear nucleate pore complex (NPC), which is made up of about a hundred different proteins, called nucleoporins. These are arranged in a circle to form a basketball basket by which the NLS protein, along with the transferred substrate, is transferred from the cytoplasm to the nucleus.

Proteins of up to about 40 kDa can be diffused into the nucleus. Proteins larger than 40 kDa enter the nucleus indirectly, requiring energy and NLS. Despite this, oligonucleotides of 10 to 25 bps (corresponding to an average of 6 kDa) have a limiting penetration capability. Oligonucleotides are hydrophilic in nature and do not readily penetrate cell membranes.

The mechanism of nuclear membrane transfer is as follows: NLS contains an importin α binding site. In the first phase of transport, NLS binds to the α, β importin heterodimer via the binding site. Importin β is responsible for transporting NLS together with the imported substrate to the cytoplasmic NPC parliaments. The system will immediately be translocated to the nuclear side of the NPC. Translation is an energy-intensive process, requiring GTP hydrolysis. The nuclear process is terminated by releasing the imported substrate in the nucleus and releasing the importod heterodimer, which in the dissociated state returns to the cytoplasm (5).

NLS is used in molecular biology to study the mediated transport or passive diffusion of substances, proteins through the nuclear envelope in eukaryotic cells. NLS fusion proteins with, e.g., GFP or EGFP are utilized, which is also discussed in patent application WO9849325.

Ubiquitin is part of a proteolytic degradation system that is important in many cell regulatory processes, such as cell cycle regulation, differentiation, apoptosis. It is also of considerable importance in the regulation of transcription and in oncogenesis.

Ubiquitinylation is one of the major non-lysosomal degradation processes of both short and long half-life proteins in mammalian cells. Specifically, it recognizes erroneous, abnormal, redundant or cellular proteins. First, the substrate binds covalently to the polybiquitin chain, then the 26S proteasome recognizes the substrate so bound by a specific signal and degrades it (6).

Ubiquitin is also used in practice to study proteasome activity. The vector encoding the ubiquitin-GFP fusion protein serves to evaluate activity based on reporter protein accumulation in living cells, which is the subject of patent application WO0181427.

In addition to ubiquitin, the FIST sequence (a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T)) is also known to be involved in protein degradation. This sequence was used in the construction of a vector containing the fusion protein and EGFP to increase EGFP turnover, whose overexpression becomes toxic to the cell. An example is patent application WO9954348, where the above-mentioned subject matter is solved.

SUMMARY OF THE INVENTION

This cellular system for testing entry of substances into nuclei is based on the construction of an expression vector encoding a sequence for the ubiquitin fusion protein, EGFP, and an amplified nuclear localization signal (Ubi EGFP NLS). Further, the present invention is based on the use of the aforementioned expression vector to create a stably transfected cell line producing the Ubi_EGFP_NLS fusion protein.

EGFP is used here as a reporter gene for its fluorescence ability. EGFP retains its functionality even after coupling to the target protein at both the C- and N-terminus. This has been used to construct a fusion protein with an amplified NLS sequence at the C-terminus of EGFP. In this case, the nuclear localization signal (derived from the SV40 T-large antigen isolated from the simian virus) will ensure the transport of EGFP to the nucleus, thereby ensuring green nuclear fluorescence under the conditions.

As mentioned in the Prior Art section, patent vectors encoding a GFP fusion protein, respectively. EGFP with NLS. Our proposal differs from the above-cited patent in particular in that the ubiquitin sequence is also included in the vector construct, the function of which is to ensure increased degradation of EGFP and thereby prevent possible toxic effects on living cells, as well as the arrangement of the amplified NLS sequence.

EGFP expression is driven by a strong eukaryotic CMV promoter, which is encoded in the pUF2 vector. With high EGFP expression, this protein can become toxic to cells and it responds by triggering an apoptotic cascade. The increased amount of EGFP protein can be regulated by the specific ubiquitin protein involved in proteolytic processes in the cell.

-2GB 295047 B6

The ubiquitin fusion protein with EGFP ensures degradation of the fusion protein and hence its faster turnover, thereby reducing the toxic effect of the oxygen radicals resulting from the formation of the EGFP active chromophore. In our proposed solution, ubiquitin is attached to EGFP at its N-terminus. The uniqueness of this design is that, unlike other patents, the sequence we present encodes not only ubiquitin and EGFP in-frame, but is also associated with the amplified NLS sequence.

The ubiquitin fusion protein, EGFP, and amplified NLS sequences, on the one hand, localize the EGFP in the nucleus and subsequently its green fluorescence, on the other hand, increase the overall turnover of EGFP and thereby reduce its possible toxic effect on cell culture.

As mentioned, the vector encoding UbiEGFPNLS is used to prepare a stable EGFP expressing line into the nucleus while increasing EGFP turnover. The cell assay system thus prepared is suitable for testing and monitoring the entry, transport of fluorescently labeled substances of different chemical nature into the nuclei of eukaryotic cells.

This system is suitable for testing the entry of unmodified and modified fluorescently labeled oligonucleotides into cell nuclei as potential gene therapeutics.

The vector constructs encoding the Ubi EGFP NLS have not yet been the subject of any patent solution.

DETAILED DESCRIPTION OF THE INVENTION

The backbone of the vector pUF2_Ubi_EGFP_NLS is the plasmid pUF2. It consists of 6252 bps, carries genes for neomycin and amplicillin resistance, the CMV promoter. The im frame gene encoding the Ubi EGFP NLS protein fusion is cloned into this plasmid by Xbal and Notl restictases.

The ubiquitin sequence was amplified by PCR. The template DNA was human cDNA. Using a DNA / RNA synthesizer (ABI 394; Applied Biosystems, Foster City, USA), primers were synthesized:

Ubisense: 5'-CGG GAT CCC CGC GCC ATC ATG

Ubiantisense: 5'- GGA ATT CGG TAC CGC GCA GAC GCA GCA

The Ubi sense primer is designed to encode a BamHI and Bblll restriction site, the primer also contains an ATG codon with a Kozak sequence. The Ubi antisense primer encodes a restriction site for KpnI and EcoRI.

The amplified DNA was digested with KpnI and BamHI and ligated with T4 DNA ligase into the pUC131 vector digested with the same restriction enzymes. 2. The recombinant plasmid pUC131_Ubi thus prepared was amplified in E. coli bacterial culture, and plasmid DNA (QUIGEN kit) was then isolated. The insert was verified by restriction and sequencing.

The ubiquitin sequence of 242 bps was further cloned with BamHi and NcoI into the pEGFP vector (Clontech) digested with the same enzymes, see FIG. 3.

The size of the plasmids pEGFP is 3355 bps. The region from 289 to 1008 bps encodes the EGFP gene. EGFP from the pEGFP vector is a modified wild-type GFP variant and is characterized by brighter fluorescence with an excitation peak of 488 nm and an emission peak of 507 nm.

The amplified NLS sequence was cloned into the resulting recombinant vector pUbi EGFP, see FIG. This 84 bps sequence is inserted from 4 partial oligonucleotides designated NLS1-4, wherein the NLS1 and NLS2 oligonucleotides form a 5-3 'strand, the NLS3 and NLS4 oligonucleotides form a complementary strand in this direction. 3'-5 '.

Using a DNA / RNA synthesizer (ABI 394), phosphorylated NLS 1-4 oligonucleotides of the following sequence were synthesized at the 5 'end:

NLS 1: 5-GTA CAT AGA TCA AAA AAA GAA GAG AAA AAA TCC AAA AAA AAA GAA AAA GGT AGA-3 '

NLS 2: 5-TCC AAA AAA AAA GAA AAA GGT ATA AGC-3 '

NLS 3: 5'-TTT TGG ATC TAC TCT CTT CTT CTT TTT TGG ATC TAT-3 '

NLS 4: 5-GGC CGC TTA TAC CTT TCT CTT CTT TTT TGG ATC TAC CTT TCT CTT CTT-3 '

These sequences are designed such that, after appropriate hybridization of mutually complementary regions (NLS 1 and 3; NLS 2 and 4), they form cohesive ends at both ends cohesive for the restriction enzymes BsrGI (5'-end) andNotI (3'-end).

NLS 1 and 2, when coupled with T4 DNA ligase, form a 5'-3 'strand, while the NLS 3 and 4 oligonucleotides form a complementary 3'-5' strand.

This designed amplified NLS sequence encodes three consecutive repeats of the GAC CCA motif AAA AAG AAG AGA AAG GTA. The sequence also encodes the TAG stup codon.

Artificial gene folding protocol:

1. In tube A, mix 200 pmoles of NLS1 and NLS3 oligonucleotides.

2. Mix 200 pmoles of NLS2 and NLS4 oligonucleotides in tube B.

3. Incubate at 80 ° C for 2 min.

4. Incubate for 30 min at room temperature.

5. Mix tube A contents with tube B and incubate for 5 min at 40 ° C.

6. Incubate for 20 min at room temperature.

7. Ligation of hybridized oligonucleotides with T4 DNA ligase (New England Biolabs) for 1 hour at 16 ° C.

The product was isolated by gel electrophoresis.

The actual cloning then involves digestion of pUbi EGFP with BsrGIaNot1 and the NLS sequence with the designed sticky ends for the two enzymes was cloned into the plasmids thus prepared. The resulting 3646 bps plasmid pUbiEGFPNLS was transformed with E. coli, multiplied under the selection pressure of the ampicillin antibiotic. Individual clones were isolated and plasmids with inserts verified by restriction.

XbaI and NotI cassettes encoding the 1043 bps Ubi EGFP NLS fusion protein were excised from pUbi EGFP NLS and inserted into the target vector pUF2 digested with the same enzymes, see FIG. 5. Insert was after vE multiplication. coli again isolated and verified by restriction.

-4GB 295047 B6

This UbiEGFPNLS cassette can be cloned into any vector by suitable restriction enzymes. For example, cloning a cassette cleaved from the pUbiEGFPNLS vector with SalI, NotI enzymes into XhoI, Not digested pLNCX2 plasmids, with SalI and XhoI having 5 compatible cohesive ends.

The vector pUF2_Ubi_EGFP_NLS was tested in the human larynx carcinoma cells (Hep2) eukaryotic cell line. Cells were cultured in DMEM 1x high glucose (L-glutamine 584 mg / l, sodium pyruvate 110 mg / l) medium (PAA Laboratories) containing 10% FCS (PAA Laboratories) and 50 µg / ml gentamycin (PAA Laboratories). Cells were cultured in an incubator at 37 ° C, 5% CO ₂ .

The day before transfection, cells were plated at a density of 4x10 ⁴ per 24-well plate. 0.4 µg of plasmids pUF2_Ubi_EGFP_NLS were transfected with Effectene. Transfection Reagent 15 (QIAGEN). 24 hours after transfection, positively transfected cells were detected by fluorescence microscopy (Nicol Eclipse TE300) using a B-2A filter block (Nikon, ex 450-490 nm, DM 505 nm, BA 520 nm). 24 hours after transfection, the cell line was subjected to selection pressure of antibiotic G418 (PAA Laboratories) at a concentration of 400 pg / ml medium for 20 days. Clones expressing EGFP were further isolated using cloning disks (Sigma).

Overview of picture documentation

Giant. 1: Reaction mechanism of GFP / EGFP active chromatography 25 Taken from: Tsien R.Y .: The Green Fluorescent Protein, Annu, Rev. Biochem., 67, 509-44 (1998) Giant. 2: Cloning scheme pUC131_Ubi 30 FIG. 3: Cloning scheme of pUbi EGFP Giant. 4: Cloning scheme of pUbi EGFP NLS Giant. 5: Cloning scheme pUF2_Ubi_EGFP_NLS

Abbreviations used in text and figures:

ampR

BGHpA bps cut EGFP GFP GTP

LacZ lITRleft neoR NL With pCMF pLac POenh pTK ampicillin resistance bovine growth hormone base pais polyadenilization signal, pairs are usually labeled with cleaved plasmids by restriction enzyme enhanced green fluorescing protein green fluorescing protein geunosine triphosphate gene for β-galactosinase promoter signaling repeat nucleotide inverted locator repeat cytomegalovirus lactose promoter polyoma enhancer thimidine kinase promoter uUC ori rITR

SD / SA intron

SV40pA

Ubi pUC plasmid replication origin right inverted terminal repeat splice donor / splice acceptor site ubiquitin polyadenlation signal

Industrial applicability

This cellular assay system can be used to test and monitor the entry, transport of fluorescently labeled substances of various chemical nature into nuclei of eukaryotic cells.

This system is suitable for testing the entry of unmodified and modified fluorescently labeled oligonucleotides into cell nuclei as potential gene therapeutics.

Reference:

(1) Dopf J. Horiagon T. M.: Deletion mapping of Aequorea victoria green fluorescent protein, Gene, 173, 39-44, 1996. (2) Tsien R.Y .: The green fluorescent protein, Annu. Roar. Biochem., 67, 509-44, 1998 (3) Hsioa-Sheng L., Ming-Shiou J. et al., Is: Green fluorescent protein toxic to the living cells, Biochem. and Biophys. Res. Communications, 260, 712-717, 1999 (4) Zimmer M .: Green Fluorescent Protein (GFP): Applications, Structure, and Related Photophysical Behavior, Chem. Rev., 102, 759-781, 2002 (5) Escriou V., Carriere M. et al .: NLS bioconjugates for targeting therapeutic genes to the nucleus, Advanced Drug Delivery Reviews, 55, 295-306, 2003 (6) Attaix D., Combaret L. et al.: Regulation of Proteolysis, Current Op. In clin. Nutrition and Metabolic Care, 4, 45-49, 2001

Claims (7)

PATENT CLAIMS Ubi EGFP NLS fusion protein of amino acid sequence _NH2 -MEIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGT $$$ MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDAT YGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRILLKGIDFKEDGNILGHKLEYNYNSHNVYIMAD KQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYI $$$ DPKKKRKVDPKKKRKVDPKKKRKV-COOH, where $ is any amino acid. 2. NLS1 to NLS4 oligonucleotides having NLS 1 sequence: 5'GTACATAGATCCAAAAAAGAAGAGAAAGGTAGATCCAAAAAAGAAGAGAAAGGTAGA-3 '; NLS 2: 5-TCCAAAAAAGAAGAGAAAGGTATAAGC-3 '; NLS 3: 5'TTTTGGATCTACCTTTCTCTTCTTTTTTGGATCTAT-3 '; NLS 4: 5-GGCCGCTTATACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTT-3 'used to fuse the amplified NLS sequence to the C-terminus of EGFP A plasmid or viral DNA or RNA vector comprising the sequence encoding the polypeptide of claim 1. A prokaryotic host cell transformed with the vector of claim 3. The eukaryotic host cell into which it has been introduced, by transfection or other means, a vector according to claim 3. Use of the plasmid or viral DNA or RNA vector according to claim 3 and / or the cells according to claim 5 for testing and monitoring the entry and / or transport of fluorescently labeled synthetic molecules of different chemical nature into the nuclei of eukaryotic cells. Use of the plasmid or viral DNA or RNA vector according to claim 3 and / or the cells according to claim 5 for testing and monitoring the entry and / or transport of fluorescently labeled synthetic molecules of different chemical nature into nuclei of eukaryotic cells, wherein the synthetic molecule is an oligonucleotide; or another molecule capable of binding to the DNA structure.