TWI385250B - Type iii promoter of rna polymerase iii - Google Patents

Description

a third type of promoter driven by a third type of RNA polymerase

The present invention is related to molecular biology.

In 1998, Fire first proposed a mechanism for double-stranded RNA-mediated specific gene silencing (Fire et al., Nature 391: 806-811, 1998), in which it was pointed out that in many organisms, exogenous or internal The double-stranded RNA (dsRNA) is introduced into the cell, and the mRNA homologous to the dsRNA is degraded, so that the corresponding gene is inhibited. This post-transcriptional gene silencing (PTGS) ) is a kind of gene suppression at the RNA level, so it is called RNA interference (RNAi).

RNAi is now widely considered to be an ancient natural cellular defense mechanism that can be used in insects or plants to combat double-stranded RNA intermediates or secondary products produced by pathogenic bacteria. However, in mammalian systems, double-stranded RNAs longer than 30 bases act directly on intracellular RNA-binding proteins, initiate type 1 interferon responses and activate non-specific RNases, resulting in Apoptosis of mammalian cells. Tuschl's team further found that the direct synthesis of 21-nucleotide double-stranded siRNA (short interfering RNA) with three-terminal prominence can specifically inhibit endogenous and exogenous gene expression in a variety of mammalian cell lines (Elbashir et Al., Nature 411: 494-498, 2001). It has been found that many mammalian gene functions are rapidly discovered and understood. Due to the importance and extensive research of RNAi in biological processes, the Science Journal declared "RNAi" a major breakthrough in 2002.

There are several different methods for driving RNAi in cells, one of which is the method of direct synthesis, but the cost of RNA oligonucleotides is higher than that of DNA oligonucleotides, and there is a problem of DNA contamination; Production of transcribed RNA oligonucleotides in vitro using T7 or other phage may have The effect is reduced, but the preparation process is complicated and still cannot solve the problem of DNA contamination. Since siRNAs are between 19-26 nucleic acids in length, it is known that such short RNAs can be driven by a type III RNA polymerase to synthesize a hairpin-like dsRNA (shRNA), whereas in mammalian cells, such shRNAs It is cleaved by RNase III (Dicer) into siRNA to further achieve gene suppression. Therefore, finding a promoter that appropriately drives the third-type RNA polymerase will be the most problem to be solved in the field of gene suppression.

The present invention is to find a third type of promoter of three type III RNA polymerases, and the novel promoter and the shRNA sequence designed according to a specific gene can be further transferred into a vector by a general molecular biological method. As a method of inhibiting a specific gene, a transgenic animal having a specific gene inhibition can also be obtained.

Thus, one aspect of the invention is a promoter, wherein the promoter is a third type of promoter that drives a third type of RNA polymerase, comprising a nucleotide sequence, wherein the nucleotide sequence It is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5.

Another aspect of the invention is a vector, wherein the vector comprises a nucleotide sequence as described above.

Another aspect of the invention is a method for promoting inhibition of the expression of a particular gene in a host cell, wherein the method comprises: (1) designing a short RNA interference oligonucleotide sequence of the particular gene; (2) After linking the forward and reverse short RNA interference oligonucleotide sequences of the designed specific gene, the vector is selected as the carrier of Patent Application No. 5; (3) after expressing the vector obtained in the step (2) in a large amount, Feeding into the host cell; (4) a vector that is introduced into the cell, and the oligonucleotide sequence which is expressed by the promoter and which interferes with the forward and reverse short RNA forms a short hairpin in the shape of a hairpin Structure (shRNA); (5) The hairpin-shaped short RNA structure can achieve the inhibition of the specific gene in the cell.

Another aspect of the invention is a genetically transformed animal, wherein the animal comprises a nucleotide sequence as described above.

Other features and advantages of the present invention will be described in more detail by the following exemplary embodiments and the accompanying drawings.

The invention will be described in more detail with reference to the following specific but non-limiting examples.

A aspect of the invention is a promoter, wherein the promoter is a third type of promoter driving a third type RNA polymerase, comprising a nucleotide sequence, wherein the nucleotide sequence is selected Free group consisting of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. Its sequence is:

SEQ ID NO: 1 (pY1):

SEQ ID NO: 3 (pY3):

SEQ ID NO: 5 (pU6):

The term "promoter" refers to a sequence of deoxyribonucleic acid (DNA) that, in genetics, enables transcription of a gene. The promoter can be recognized by RNA polymerase and transcription begins. Usually the transcription start point will be numbered starting from +1. Since the promoter is generally upstream of the gene, its number is a negative number of +1 inverse number, for example -100 is the upstream base pair from the starting point position 100. Eukaryotic promoters are very diverse and difficult to have a clear feature, but there are still areas that are shared together. For example, most eukaryotic promoters, but not all, contain a TATA box (sequence TATAAA) that binds to TATA binding proteins to aid in the formation of RNA polymerase transcription complexes.

RNA polymerase is an enzyme responsible for the production of RNA from DNA templates. This process is called transcription. RNA polymerase is a very important enzyme found in all living organisms and in many viruses. In eukaryotes, RNA polymerase can be divided into three types: first-type RNA polymerase synthesizes ribosomal RNA (rRNA), second-type RNA polymerase synthesizes messenger RNA (mRNA), and type III RNA polymerase synthesizes transporter. RNA (tRNA), rRNA5s and other short RNAs that can be found in cells.

Promoters for driving type III RNA polymerase can be divided into three categories. The first and second types of promoters contain a CREs region (cis-regulatory elements) located downstream of the transcription start position, but all third classes. The CREs region of the promoter is located upstream of the transcription start position (Murphy, S. et al., Mol Cell Biol 12: 3247-61, 1992). A typical third-type promoter sequence is currently known to contain a DSE sequence (Distant Sequence Element) located approximately -245 to -210 base positions upstream of the transcription start position, which contains an Oct-1 binding site, and a The SPH position, a PSE sequence (Proximal Sequence Element) is located approximately -65 to -48 bases upstream of the transcription start position, and a TATA box.

The three promoter sequences of the present invention are shown in SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, respectively, or a functional or sequence variant thereof. Among them, among the above promoter sequences, there are several regions with high retention, such as PSE ( STSAC CGTGW STGTR AARTG ), Oct (Octamer) ( ATTTG CAT, or CTGCAAAT ), and SPH (SphI postoctamer homology) (Zamord, Z. And Stumph, WE, Nucleic Acids Res, 18: 7323-7330, 1990) (YYWCC CRNMA TSCMY YRCR) and other areas.

The term "Y" refers to thymine/uracil or cytosine.

The term "S" refers to guanine or cytosine.

The term "W" refers to adenine or thymine/uracil.

The term "R" refers to 嘌呤.

The term "M" refers to adenine or cytosine.

The term "N" refers to any base.

The term "functional or sequence variant" refers to one and SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 having more than 80% in the highly reserved region, and 50 in the non-highly retained region. A nucleotide sequence having a % similarity and a nucleotide sequence having the function of a promoter of a third type RNA polymerase. This variant may be derived from a mutation of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5, for example, at a position that does not affect promoter activity. Those skilled in the art can readily use general molecular biology methods to infer the location of nucleotide sequences that are related or unrelated to promoter activity, for example, using point mutation analysis. It is further possible to use bases of similar chemical nature to be substituted or deleted without affecting the activity of the promoter. Any of the variants of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 are also expected to retain their activity to drive expression of the third type RNA polymerase. Variants on this sequence can be tested for activity by the method of Example 3.

Preferably, the three promoter sequences of the present invention are sequenced as in Example 1, respectively. The results are shown in SEQ ID NO: 2, SEQ ID NO: 4 and SEQ ID NO: 6. The sequences are:

SEQ ID NO: 2 (pY1)

SEQ ID NO: 4 (pY3)

SEQ ID NO: 6 (pU6)

The three promoter sequences found in the present invention can be further transformed into a vector in. Thus, another aspect of the invention is a vector wherein the vector comprises the promoter sequences of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 as described above. The promoter is ligated into the vector, and the person skilled in the art can design it according to the general molecular biology method. The source of the carrier can be manufactured by a person skilled in the art or from a commercially available carrier. According to an embodiment of the invention, the vector is a pSUPER-GFP-1-neo vector which is GFP and by GSU-Gly-Ser-Gly-Gly pentapeptide from pSUPER-GFP/neo (OligoEngine, Inc., USA) Neo is linked to a complex protein modification. The vector can also be inserted into a shRNA sequence of a specific gene designed or disclosed by a person skilled in the art. When the vector is introduced into a living cell, the expression of the third type RNA polymerase can be driven to transcribe the shRNA to reach the subsequent gene. The purpose of inhibition.

Thus, another aspect of the invention is a method of promoting inhibition of the expression of a particular gene in a host cell, wherein the method comprises: (1) designing a short RNA interference oligonucleotide sequence of the particular gene; 2) linking the forward and reverse short RNA interference oligonucleotide sequences of the designed specific gene to the vector of Patent Application No. 5; (3) after expressing the vector obtained in the step (2) in a large amount, Carrying it into the host cell; (4) a vector that is introduced into the cell, and the oligonucleotide sequence which is expressed by the promoter and which interferes with the forward and reverse short RNA forms a hairpin shape. A short RNA structure (shRNA); and (5) a short RNA structure of the hairpin shape can achieve an effect of inhibiting the specific gene in the cell.

The term "host cell" refers to a receptor that is received or received into a vector comprising the promoter of the present invention, which is a living cell. According to current research, promoters of type III RNA polymerases from prokaryotes to eukaryotes have similar sequences in highly retained regions (Wray, GA et al., Mol Biol Evol 20: 1377-1419, 2007), therefore, the promoter of the present invention can be expressed in cells of animals, plants or protozoa. A preferred embodiment is for expression in animal cells and is selected from the group consisting of humans, pigs, cows, sheep, horses, rats, rabbits, chickens, ducks and geese. According to an embodiment of the invention, it is expressed in cells of humans and pigs.

Still another aspect of the invention is a gene-transforming animal, wherein the animal line comprises a nucleotide sequence as described above. Preferably, the genetically transformed animal is selected from the group consisting of pigs, cows, sheep, horses, rats, rabbits, chickens, ducks and geese.

The invention will be further illustrated by the following examples, but the actual invention is not limited by the examples shown.

Example 1: Sequence of a promoter of the third type of RNA type 3 polymerase of sequencing pigs

The Genome walking method is an important molecular biology research technique. Using a known gene or molecular marker to perform a polymerase chain reaction continuously, an unknown gene sequence adjacent to a known gene can be obtained. This method can be used. Search and identification of promoter sequences.

The experiment used the DNA Walking SpeedUp kit (SeeGene Inc., USA) as a tool for finding promoters. First, the primers were designed based on the promoter sequences of the human and mouse four-stage promoters -Y1 (hY1, mY1), Y3 (hY3, mY3), U6 (hU6, mU6) and 7SK (h7SK, m7SK). (As in Table 1), the above sequence was used to perform a three-polymerase chain reaction with a gene template from Duroc pig. The three polymerase chain reaction conditions are as follows: first reaction:

Second reaction:

The third reaction:

The results of electrophoresis of the product of the third polymerase chain reaction are shown in Figure 1. The DNA product indicated by the arrow in Fig. 1 was taken out from the colloid and transferred to pGEM T-Easy Vector (Promega Corporation, USA). Perform the sequencing work.

Example 2: Promoter sequence analysis

The sequence of the sequence obtained in Example 1 was compared with the promoter sequences of pY1, pY3, pU6 and p7SK corresponding to human and mouse, respectively, and the alignment results are shown in Figures 2A-D.

As shown in Figure 2A, the pY1 promoter (SEQ ID NO: 2) consists of a promoter element of the third type of a typical third-type RNA polymerase, including a TATA box of -31 to -25 base positions. The PSE at the -67 to -48 base position, the SPH at the -217 to -199 base position, and the Oct at the -231 to -223 base position. The bottom line in Fig. 2A indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is a nucleotide sequence which is substituted according to the corresponding promoter of other species of animals. After the alignment, bases replaceable in the highly reserved region were deduced, and the sequence thereof is shown in SEQ ID NO: 1.

As shown in Figure 2B, like the pY1 promoter, the pY3 promoter (SEQ ID NO: 4) consists of a TATA box at the -31 to -25 base position, located at the -69 to -45 base position PSE, located at SPH at positions -246 to -228, and Oct at positions -259 to -252. The pY3 promoter also contains a CACCC region located at -245 to -241, which has been found in both bovine and human 7SK (b7SK and h7SK) and is predicted to enhance promoter activity (SC Bannister et al) ., RMC Biotechnol 7:79, 2007). The bottom line in Fig. 2B indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is the nucleotide sequence which is substituted according to the corresponding promoter of other species of animals. After the alignment, bases replaceable in the highly reserved region were deduced, and the sequence is shown in SEQ ID NO: 3.

As shown in Figure 2C, the pU6 promoter (SEQ ID NO: 6) is like the U6 promoter of human (hU6) and murine (mU6), both of which are located in the TATA box of -31 to -25 base positions. The PSE at positions of 68 to -51 bases, Oct at positions of -230 to -223 bases, and SPH at positions of -249 to -231 bases. Different from the start of Y1, Y3 and 7SK The subsequence, the Oct of the pU6 promoter, is located in the downstream region of the SPH. The bottom line in Fig. 2C indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is the nucleotide sequence which is substituted according to the corresponding promoter of other species of animals. After the alignment, bases replaceable in the highly reserved region were deduced, and the sequence is shown in SEQ ID NO: 5.

As shown in Figure 2D, the promoter of p7SK (SEQ ID NO: 8) consists of a TATA box at the -31 to -25 base position, a PSE at the base position of -66 to -47, at -226 to - SPH at 208 base positions, and Oct at -242234 base positions. The upstream region of the 5' end of purified p7SK is consistent with the endonuclease/reverse enzyme region contained in porcine chromosome 7. The bottom line in Fig. 2D indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is a nucleotide sequence which is replaceable according to the corresponding promoter of other species of animals. After the alignment, bases replaceable in the highly reserved region were deduced, and the sequence is shown in SEQ ID NO: 7.

Example 3: Construction of a expression vector for a short RNA structure (shRNA) having a hairpin shape

According to the upstream region of the 5' end of Y1, Y3, U6 and 7SK, a forward primer comprising an AatII restriction enzyme cleavage site and a reverse primer comprising a BglII restriction enzyme cleavage site were designed, respectively, and the sequences thereof are shown in Table 2. Each 0.4 μM forward and reverse primer was added to the polymerase chain reaction solution together with 0.1 μg of Duroc porcine genomic DNA template at 95 ° C for 2 minutes; then 94 ° C for 30 sec / 58 ° C for 40 sec / 72 32 cycles were carried out at ° C for 30 seconds; finally, at 72 ° C for 7 minutes.

The product fragment of the polynuclease chain reaction cleavage by AatII and BglII was cloned into the pSUPER-GFP-1-neo vector, wherein the GFP-1-neo complex gene of the vector recorded GFP linked by five amino acids of GGSGG. Fusion protein with neo (Brummelkamp, TR et al., Science, 296: 550-553, 2002). The vector completed after colonization is shown in Figure 3, wherein the sequence inserted at the position of Pol III promoter in Figure 3 is the product fragment which is cleaved by AatII and BglII after polymerase chain reaction with the primer of Table 2, and The completed shRNA expression vectors were named ppY1-MGS, ppY3-MGS, ppU6-MGS and pp7SK-MGS, respectively.

Example 3: Analysis of promoter activity

In order to analyze the activity of the promoter, this experiment is to simultaneously deliver the shRNA expression vector and the vector carrying the luminescent enzyme into a living cell, and use the expression amount of luminescence as an index for suppressing the expression of the cold light gene by the shRNA vector. The lower the amount of cold light expression, the stronger the gene inhibition effect, and the stronger the activity of the promoter.

First, shRNAs designed to inhibit the luciferase gene were designed. In this experiment, a 21-base short sequence was designed for the luminescent enzyme, and its sequence was AAGAT CCGCG AGATT CTCAT T (SEQ ID NO: 31), which was named Luc2#3. Next, a forward sequence comprising the XhoI restriction enzyme cleavage site (SEQ ID NO: 29) and a reverse sequence comprising the BglII restriction enzyme cleavage site (SEQ ID NO: 30) were further designed as shown in Table 2. After combining the two segments, they were fed into the shRNA expression vectors of ppY1-MGS, ppY3-MGS, ppU6-MGS and pp7SK-MGS, respectively, and the efficiency of shRNA production by each promoter was observed.

1 μg of the vector pGL4.13 (Promega Corporation, USA) with luciferase and 1.5 μg of shRNA expression vector with shRNA that inhibits the luciferase gene The preparation vector (shRNA containing no Luc2#3) was first diluted with 100 μl of 0.15 M sodium chloride solution, added with 8 ul of ExGen500 (Fermentas International Inc., Canada), and simultaneously infected and fed into porcine embryonic kidney epithelial cell line LLC. -PK1 and human embryonic kidney epithelial cell line HK293. After 48 hours into the cells, the cells were decomposed with 500 μl of Glo Lysis Buffer (Promega Corporation, USA), and 50 μl of Bright-Glo (Promega Corporation, USA) reagent was added to 50 μl of cell decomposing material in a 96-well microplate to GloMax luminescence ( The Promega Corporation, USA) system measures the activity of luminescent enzymes.

As shown in the results of Fig. 4, when 1.5 μg of the shRNA expression vector carrying the shRNA which inhibits the luminescent enzyme gene was added, ppY1-MGS-Luc2#3, ppY3-MGS-Luc2#, except that pp7SK-MGS had no significant inhibitory effect. 3, and ppU6-MGS-Luc2#3 have about 88%, 82%, and 94% inhibition for luminescent activity, respectively. From the results of the porcine embryonic kidney epithelial cell line, ppU6-MGS-Luc2#3 was the best. As shown in Fig. 5, in the human embryonic kidney epithelial cell line, the same inhibitory effect was observed when the same experiment was carried out as the shRNA expression vector of ppU6-MGS-Luc2#3.

Therefore, as can be seen from the results of Fig. 4 and Fig. 5, the promoters of pY1, pY3 and pU6 of the present invention all have the activity of inhibiting gene expression, and the cells of human or pig can maintain their activity.

Those skilled in the art will be able to make changes and modifications in accordance with the embodiments without departing from the spirit of the invention. It is to be understood that the invention is not to be limited by the scope of the embodiments disclosed herein,

The foregoing description and the embodiments may achieve better illustrative effects by the additional drawings. To enhance the description of the invention, the drawings of the appropriate embodiments are set forth herein. It is to be noted that the present invention is not limited to the descriptions set forth herein.

Figure 1 shows the electrophoresis of the third polymerization acidase reaction product by chromosome walking method. Figure. The position indicated by the white arrow is the position at which the colloid treatment is taken out.

Figure 2A is a graph showing the results of alignment of pY1 and mY1 sequences. The bottom line indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is a nucleotide sequence which is replaceable according to the corresponding promoter of other species.

Figure 2B is a graph showing the results of alignment of pY3 with mY3 sequences. The bottom line indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is a nucleotide sequence which is replaceable according to the corresponding promoter of other species.

Fig. 2C shows the results of alignment of pU6 with mU6 and hU6 sequences. The bottom line indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is a nucleotide sequence which is replaceable according to the corresponding promoter of other species.

Figure 2D is a graph showing the results of alignment of p7SK with h7SK sequences. The bottom line indicates a highly reserved region, the nucleotide sequence on the bottom line is the sequence obtained by sequencing, and the bottom line is a nucleotide sequence which is replaceable according to the corresponding promoter of other species.

Fig. 3 is a view showing the construction of a carrier. The position of the PoIIII promoter is the position at which the promoter of the present invention is placed.

Figure 4 is a graph showing the ability of shRNA expression vectors comprising the promoter of the present invention to inhibit cold light gene expression in porcine embryonic kidney epithelial cells.

Figure 5 is a graph showing the ability of shRNA expression vectors comprising the promoter of the present invention to inhibit cold light gene expression in human embryonic kidney epithelial cells.

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Claims (9)

a promoter, wherein the promoter is a promoter of a third type that drives a third type RNA polymerase, comprising a nucleotide sequence, wherein the nucleotide sequence is selected from SEQ ID NO: 1 a group consisting of SEQ ID NO: 3 and SEQ ID NO: 5. The promoter of claim 1, wherein the nucleotide sequence of the promoter is the sequence of SEQ ID NO: 2. The promoter of claim 1, wherein the nucleotide sequence of the promoter is the sequence of SEQ ID NO: 4. The promoter of claim 1, wherein the nucleotide sequence of the promoter is the sequence of SEQ ID NO: 6. A vector, wherein the vector comprises the nucleotide sequence of claim 1. A method for promoting inhibition of the expression of a particular gene in a host cell in vitro, wherein the method comprises: (1) designing a short RNA interference oligonucleotide sequence of the particular gene; (2) designing a specific gene positive After ligating to the oligonucleotide sequence which interferes with the reverse short RNA, the vector is selected as in the scope of claim 5; (3) after expressing the vector obtained in the step (2) in a large amount, it is sent to the host cell; (4) a vector which is introduced into a cell, and the oligonucleotide sequence which is expressed by the promoter and which interferes with the forward and reverse short RNA forms a short RNA structure (shRNA) in the shape of a hairpin; 5) The hairpin-shaped short RNA structure can achieve the inhibition of the specific gene in the cell. The method of Patent Application No. 6, wherein the host cell line is an animal cell, a plant cell or a protozoan. The method of claim 6, wherein the host cell line is an animal cell. The method of claim 8, wherein the animal is selected from the group consisting of human, pig, cow, sheep, horse, mouse, rabbit, chicken, duck and goose.