CZ200475A3 - 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

Universal cell system for testing the entry of substances into nuclei

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- termini while maintaining fluorescence.

GFP fluoresces due to the chromophore that is formed autocatalytically by cyclizing three amino acids in the presence of molecular oxygen. It does not require any other substrates or coactors 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 produced with active chromophore (2). 1. Generated oxygen radicals become toxic to the cell 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 (enhanced GFP) is a mutant wild-type variant of GFP in which two amino acids have been substituted, namely a Phe to Leu substitution at position 64 and a 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 the wild type fluoresces at room and temperature

* -4 - * lower, 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 use of a GFP fusion protein, e.g., with a Gas protein, is patent

Z5i ^ zVo03008435.

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 a nuclear pore complex (NPC) consisting 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 mediately, requiring energy and NLS.

Despite this, oligonucleotides of 10 ^ 25 bps (corresponding to an average of 6 kDa) have a limiting penetration capability. Oligonucleotides have a hydrophilic character and do not readily penetrate cell membranes.

The mechanism of nuclear membrane transfer is as follows: NLS contains a binding site for portportin a. In the first phase of transport, the NLS binds to the α, β importin heterodimer via said binding site. portportin β is responsible for transporting the NLS along with the imported substrate to the cytoplasmic NPCs. The system will immediately be translocated to the nuclear side of the NPC. Translocation is an energy-intensive process, requiring GTP hydrolysis. The transnuclear process is terminated by releasing the imported substrate in the nucleus and releasing the importin 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 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 also has considerable significance. in transcription regulation and oncogenesis.

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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 polyubiquitin chain, then the 26S proteasome recognizes the substrate thus bound by a specific signal and degrades it (6).

Ubiquitin is also used in practice to study proteasome activity. The vector encoding ubiquitin-GFP fusion protein is used to evaluate activity based on reporter protein accumulation in living cells, as described in patent / WO018112277

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 an EGFP fusion protein to ₍ increase EGFP turnover, whose overexpression becomes toxic to the cell. An example is the patent where the aforementioned subject matter is addressed.

SUMMARY OF THE INVENTION:

This cellular system for testing the 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). Furthermore, the present invention is based on the use of the aforementioned expression vector to generate a stably transfected cell line producing the Ubi_EGFP_NLS fusion protein.

EGFP is used here as a reporter for its fluorescence ability. EGFP retains its functionality even after coupling to the target protein at both the C- and N-terminus. This was 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, patented vectors encoding the GFP fusion protein, respectively, are disclosed. EGFP sNLS. 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 the cell 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.

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 an 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 Ubi_EGFP_NLS 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.

Vector constructs encoding EJbi_EGFP_NLS have not been the subject of any patent solution yet.

Example 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 ampicillin resistance, the CMV promoter. An in-frame gene encoding the Ubi _EGFP_NLS fusion protein is cloned into this plasmid by XbaI and NotI.

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:

Ubi_sense: 5‘-CGG GAT CCC CGC GCC ATC ATG GAG ATC TTC GTG AAG ACC-3 bi Ubi_antisense: 5‘-GGA ATT CGG TAC CGC GCA

The Ubi_sense primer is designed to encode a BamHI and BglII restriction site, the primer also contains an ATG codon with a Kozak sequence. Primer Ubi_antisense 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 expanded in E. coli bacterial culture, and plasmid DNA (QIAGEN 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 plasmid 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 consists of 4 sub-oligonucleotides designated NLS 1-4, wherein the NLS1 and NLS2 oligonucleotides form a 5'-3 'strand, while the NLS3 and NLS4 oligonucleotides form a 3'-5 complementary strand. '.

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

NLS 1: 5‘-GTA CAT AGA TCC AAA AAA GAA GAA AAA GGT AGA TCC AAA AAAAA GA AAA GGT AGA-3

NLS 2: 5‘-TCC A AAA A GAA GAG A AAA GGT ATA AGC-3 AT

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

NLS 4: 5‘-GGC CGC TTA TAC CTT TCT CTT TCT 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 ends at both ends cohesive for the restriction enzymes BsrGI (5 'end) and Nati (3' end).

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

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

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 the cleavage of pUbi_EGFP by BsrGI and NotI and the NLS sequence with the designed sticky ends for the two enzymes was cloned into the plasmid thus prepared. The resulting 3645 bps plasmid pUbi_EGFP_NLS was transformed into E. coli, amplified under the selection pressure of an ampicillin antibiotic. Individual clones were isolated and plasmids with inserts verified by restriction.

The 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 as shown in FIG. 5. After insertion in E. coli, the insert was again isolated and verified by restriction.

This Ubi_EGFP_NLS cassette can be inserted into any vector by suitable restriction enzymes. An example would be cloning of the cassette cleaved from the vector pUbi_EGFP_NLS by SalI, NotI enzymes into XhoI digested plasmid pLNCX2, NotI, with SaU and XhoI having mutually 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), 50 µg / ml gentamycin (PAA Laboratories). Cells were cultured in an incubator at 37 ° C, 5% CO 2.

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The day before transfection, cells were plated at a density of 4x10 ⁴ per 24-well plate. 0.4 µg of plasmid pUF2_Ubi_EGFP_NLS was transfected with Effectene Transfection Reagent (QIAGEN). 24 hours after transfection, positively transfected cells were detected by fluorescence microscopy (Nikon 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).

Picture documentation:

Giant. 1: Reaction mechanism of active chromophore GFP / EGFP, taken from: Tsien R.Y .: The green fluorescent protein, Annu. Roar. Biochem., 67, 509-44 (1998)

Giant. 2: Cloning scheme of pUC131_Ubi

Giant. 3: Cloning scheme pUbi_EGFP

Giant. 4: Cloning scheme pUbi_EGFP_NLS

Giant. 5: Cloning scheme of pUF2_Ubi_EGFP_NLS

Abbreviations used in text and figures: ampR ampicillin resistance BGHpA bovine growth hormone polyadenilation signal bps base pairs cut restriction enzyme digestion of the digested plasmid EGFP enhanced green fluorescent protein GFP green fluorescent protein GTP guanosine triphosphate LacZ β-galactosidase gene 1ITR left inverted terminal repeat neoR neomycin resistance NLS nuclear localization signal pCMV a cytomegalovirus-derived promoter cry a lactose promoter POenh polyoma enhancer pTK thimidine kinase promoter pUC ori pUC plasmid replication origin rITR right inverted terminal repeat SD / SA intron splice donor / splice acceptor site SV40pA polyadenilation signal Ubi ubiquitin

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Industrial Applicability:

This cellular assay system can be used to test and monitor the entry, transport of fluorescently labeled substances of different 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) Hsiao-Sheng L., Ming-Shiou J. et al., Green fluorescent protein toxic to the living cells? 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 .: Reguiation of Proteolysis, Current Op. In clinic nutrition and metabolic care, 4, 45-49, 2001

Claims (7)

Claims: 1. Ubi_EGFP_NLS amino acid sequence / NH2 MEIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLED GRTLSDYN1QKESTLHLVLRLRGT $$$ MVSKGEELFTGVVPILVELDGDVNGH KFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHM KQHDFFKSAMPEGYVQERTJFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFK EDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQ QNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAG1TLGMDEL YI $$$ DPKKKRKVDPKKKRKVDPKKKRKV -COOH where $ is any amino acid. 2. NLS1 to NLS4 oligonucleotides having NLS 1 sequence: 5'-GTACATAGATC CAAAAAAGAAGAGAAAGGTAGATCCAAAAAAG AAGAGAAAGGTAGA-3 ⁴ ; NLS 2: 5'-TCCAAAAAAGAAGAGAAAGGTATAAGC-3 '; NLS 3: 5'-TTTT GGATCTACCTTTCTCTTCTTTTTTGGATCTAT- ³⁴ ; NLS 4: 5'-GGCCGCTTA TACCTTTCTCTTCTTTTTTGGATCTACCTTTCTCTTCTT-3 ⁴ 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. A eukaryotic host cell into which transfection or other means has been introduced. Use of a plasmid or viral DNA or RNA vector according to claim 3 and / or 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 a plasmid or viral DNA or RNA vector according to claim 3 and / or 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 other molecule capable of DNA structure.