Patent Application
20230270087
This patent application claims the benefit and priority of Chinese Patent Application No. 202210191036.7 filed with the China National Intellectual Property Administration on Feb. 25, 2022, the disclosure of which is incorporated by reference herein in its entirety as part of the present application.
A computer readable XML file entitled “GWP20220801139”, that was created on Dec. 13, 2022, with a file size of about 28,826 bytes, contains the sequence listing for this application, has been filed with this application, and is hereby incorporated by reference in its entirety.
The present disclosure belongs to the technical field of bioengineering, and relates to a method for constructing a Ptgds gene knockout rat model with spontaneous kidney yin deficiency.
Perimenopause generally refers to a natural menopausal state after the depletion of follicles in a women's ovaries. It usually shows various symptoms related to the “heart-kidney-reproductive”axis, including symptoms of hot flashes (or vasomotion), chronic renal disease, cardiovascular disease, and neurodegenerative disease, etc. These physical or psychological symptoms are called perimenopausal syndrome. Long-term studies have shown that the perimenopausal syndrome is closely related to development of the chronic kidney disease. From the perspective of traditional Chinese medicine dialectics, the perimenopausal syndrome is due to kidney deficiency, mostly kidney yin deficiency, typically showing symptoms of hot flashes, obesity, and central degeneration. Perimenopausal syndrome is due to the fact that from the age of 49, kidney qi gradually becomes deficient in women, and the effect of menstruation also disappears. Conception Vessel and Thoroughfare Vessel tend to be feeble, and the essence and blood for nourishing yin decrease, resulting in symptoms of yin deficiency, amenorrhea, and gradual infertility. Therefore, “Su Wen” recorded: “At the age of 49, the Conception Vessel and the Thoroughfare Vessel is feeble, the menstruation is exhausted to cause amenorrhea, vagina atrophy, female characteristics are destroyed and women become infertility”.
Existing studies have shown that estrogen depletion attenuates adipocyte transport in menopausal rat models. Prostaglandin D2 synthase (Ptgds) has increased expression in kidney and decreased expression in uterus and hypothalamus, thereby weakening renal lipid metabolism. As a result, typical symptoms of kidney yin deficiency in menopause appear in a cascade, including renal metabolic disorders such as hot flashes, weight gain, elevated blood glucose, and abnormal lipid metabolism. Experimental data further confirm that upstream estrogen receptor β (ERβ) depletion activates the overexpression of Ptgds in renal, leading to an imbalance in renal lipid metabolism and the reduced transport of Ptgds to the hypothalamus. Moreover, this factor may continue to accelerate the degeneration of central nervous system function, and typical central degenerative symptoms such as decreased learning ability and memory decline appear in the experiment.
Ptgds is a non-glutathione-independent and lipocalin-type PGD synthetase. As a monomeric member of the lipocalin family, Ptgds, is an approximately 26 kDa protein composed of 189 amino acid residues, includes a signal sequence (aa 1-24), and a lipocalin region that serves as both a catalytic site and a hydrophobic molecule transporter (aa 40-187). Ptgds is mainly localized in the Golgi apparatus and nuclear membrane of cells or secreted to extracellular regions, and are mainly expressed in the brain, central nervous system, prostate, uterus, and kidney. Ptgds has the function of catalyze the synthesis of prostaglandin D2 (PGD2) and transport the lipophilic substances. Ptgds can also catalyze the conversion of PGH2 to PGD2, thereby affecting sleep and body temperature. In addition, activation of Ptgds can affect lipid metabolic shifts, such as eicosanes metabolism in the arachidonic acid, α-linolenic acid (ala), and cyclooxygenase (cox) pathways. Urine-secreted Ptgds is synthesized in glomeruli and glomerular rings. Due to a low molecular weight and anionic properties, Ptgds can pass through the glomerular capillary wall more easily than serum albumin to more accurately reflect changes in glomerular permeability, which is the key marker for the diagnosis of kidney diseases.
In order to conduct related researches on menopausal kidney yin deficiency, animal models are generally prepared by surgically removing both ovaries of female rats. This is because the rat has a well-developed pituitary-adrenal function and a sensitive stress response, such that the rat is especially suitable for stress response and endocrine experimental researches of pituitary, epinephrine, and ovary, etc. However, the construction of rat models have several problems, such as the construction takes a long time and the model animals are difficult to be raised. Therefore, it is urgently needed to find an efficient and stable method to construct the animal models. Currently, gene knockout models are commonly constructed and bred to solve the above problem. However, there are only reports on mouse models with Ptgds gene knockout (Ptgds−/−) in the prior art, and there is no relevant report on rat models with the Ptgds−/−.
Aiming at the technical problems in the existing construction of gene knockout rat models, the present disclosure provides a method for constructing a Ptgds gene knockout rat model with spontaneous kidney yin deficiency. The method has high accuracy of gene modification, targeting specificity, and a short experimental period. Therefore, a reliable and stable genetically-engineered model is developed to lay the foundation for a therapeutic effect of menopausal syndrome-related diseases.
To achieve the above objective, the present disclosure adopts the following technical solution:
The present disclosure provides a method for constructing a Ptgds gene knockout rat model with spontaneous kidney yin, including the following steps:
1) designing two target sequences Ptgds-sgRNA1 and Ptgds-sgRNA2 at a Ptgds gene locus;
2) obtaining purified Cas9mRNA, purified Ptgds-sgRNA1, and purified Ptgds-sgRNA2 by in vitro transcription;
3) conducting targeted knockout on a 2,944 bp sequence fragment in the Ptgds gene using a CRISPR/Cas9 system to obtain a Ptgds knockout gene;
4) injecting the purified Cas9mRNA, the purified Ptgds-sgRNA1, the purified Ptgds-sgRNA2, and the Ptgds knockout gene into rat embryos, and transplanting the embryos into fallopian tubes of surrogate recipient rats to obtain neonatal rats;
5) conducting gene identification on the neonatal rats to select heterozygous rats; and
6) conducting breeding on the heterozygous rats with wild-type rats for multiple generations, and subjecting offspring rats obtained from each generation to gene identification until obtaining homozygous rats,that is the Ptgds gene knockout rat model.
Further, in step 1), the Ptgds-sgRNA1 has a nucleotide sequence set forth in SEQ ID NO: 1; and the Ptgds-sgRNA2 has a nucleotide sequence set forth in SEQ ID NO: 3.
Further, in step 3), the sequence fragment includes an intron sequence fragment and an exon sequence fragment.
Further, in step 3), the intron sequence fragment has a nucleotide sequence set forth in SEQ ID NO: 5; and the exon sequence fragment has a nucleotide sequence set forth in SEQ ID NO: 6.
Further, the gene identification in step 5) and step 6) includes the following steps:
S1) extracting a genomic DNA from the neonatal rat;
S2) conducting PCR amplification with specific primers using the genomic DNA as a template to obtain an amplification product;
S3) conducting electrophoresis detection on the amplification product using agarose gel; and
S4) identifying the heterozygous rats or the homozygous rats according to an electrophoresis result.
Further, in step S2), the specific primers include primers of Ptgds-L-S, Ptgds-L-A, Ptgds-R-S, and Ptgds-R-A, with nucleotide sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.
Further, in step S2), a reaction system of the PCR amplification includes: 2.5 μl of a template DNA at 500 ng/μl, 2.5 μl of each of the Ptgds-L-S, the Ptgds-L-A, the Ptgds-R-S, and the Ptgds-R-A that are at 10 μmol/L, 5 μl of a 10× buffer, 5 μl of dNTP at 2.5 mmol/L, 0.5 μl of Eazy-taq, and supplementing to 50 μl with water; and
the PCR amplification includes: pre-denaturation at 98° C. for 2 min; denaturation at 98° C. for 20 s×30; annealing at 55° C. for 20 s×30; extension at 72° C. for 10 s×30; terminal extension at 72° C. for 5 min; and cooling at 16° C. for 2 min.
Further, step 6) specifically includes the following steps:
6.1) using the heterozygous rats selected in step 5) as F0-generation heterozygous rats, caging with the wild-type rats, conducting gene identification on obtained offspring I, and selecting heterozygous rats from the offspring I as F1-generation rats;
6.2) caging the F1-generation rats with the wild-type rats, conducting gene identification on obtained offspring II, and selecting heterozygous rats from the offspring II as F2-generation rats; and
6.3) using rats generated by conducting self-breeding within the group of F2-generation rats as F3-generation rats, and conducting gene identification to select homozygous rats in F3-generation as the Ptgds gene knockout rat model.
The present disclosure has the following beneficial effects:
1. The present disclosure provides a CRISPR/Cas9 technology to construct a Ptgds gene knockout rat model, which has the advantage of high gene modification accuracy, specific targeting, short experimental period, and no species restriction.
2. In the present disclosure, during construction of the gene knockout rat model, nicks are made on exon 1 of the Ptgds gene spliceosome Ptgds-201 and the non-coding region of exon 7, the two nicks are directly ligated through an NHEJ repair pathway, and the sequence between the two nicks (namely the entire coding sequence) is deleted, so as to knock out the Ptgds gene. The purified Cas9mRNA and purified sgRNA obtained by in vitro transcription are injected into SD rat embryos. After injection, the embryos are transplanted into the fallopian tubes of surrogate recipient rats. And the Ptgds gene knockout rat model is obtained by breeding the embryos. The present disclosure provides a reliable and stable genetic engineering model for further researches on an influence of the spontaneous menopausal kidney yin deficiency in rats, and lays the foundation for clarifying a therapeutic effect of menopausal syndrome-related diseases and the like.
3. The present disclosure is based on use of the CRISPR/Cas9 technology in targeted knockout of the Ptgds gene in rats to construct a Ptgds gene knockout (Ptgds−/−) rat model, which has spontaneous kidney yin deficiency symptoms; through reproduction, PCR identification, and pathological index detection, the model can be further applied to pharmacodynamic evaluation. The model can provide a reliable and stable genetic engineering model for the study of symptoms of kidney yin deficiency during perimenopausal, and lay a foundation for exploration of perimenopausal syndrome mechanism and evaluation of related drug treatment effects.
The method for constructing model in present disclosure is further described in detail below with reference to the accompanying drawings and examples.
The present disclosure provides a method for constructing a Ptgds gene knockout rat model with spontaneous kidney yin deficiency, including the following steps:
1) two target sequences Ptgds-sgRNA1 and Ptgds-sgRNA2 at a Ptgds gene locus is designed.
2) purified Cas9mRNA, purified Ptgds-sgRNA1, and purified Ptgds-sgRNA2 were obtained by in vitro transcription.
3) CRISPR/Cas9 system to conduct targeted knockout on a 2,944 bp sequence fragment in the Ptgds gene to obtain a Ptgds knockout gene.
4) The purified Cas9mRNA, the purified Ptgds-sgRNA1, the purified Ptgds-sgRNA2, and the Ptgds knockout gene are injected into rat embryos, and the embryos are transplanted into fallopian tubes of surrogate recipient rats to obtain neonatal rats.
5) Gene identification was conducted on the neonatal rats to select heterozygous rats.
6) Multi-generation reproduction between heterozygous rats and wild-type rats was conducted, and offspring rats obtained from each generation are subjected to gene identification until homozygous rats are genetically identified as the Ptgds gene knockout rat model.
In the present disclosure, in step 1), the Ptgds-sgRNA1 has a nucleotide sequence set forth in SEQ ID NO: 1; and the Ptgds-sgRNA2 has a nucleotide sequence set forth in SEQ ID NO: 3.
In the present disclosure, in step 3), the sequence fragment includes an intron sequence fragment and an exon sequence fragment.
In the present disclosure, in step 3), the intron sequence fragment has a nucleotide sequence set forth in SEQ ID NO: 5; and the exon sequence fragment has a nucleotide sequence set forth in SEQ ID NO: 6.
In the present disclosure, the gene identification in step 5) and step 6) includes the following steps:
S1) a genomic DNA is extracted from the neonatal rat.
S2) PCR amplification was conducted with specific primers using the genomic DNA as a template to obtain an amplification product.
S3) electrophoresis detection was conducted on the amplification product using agarose gel.
S4) the heterozygous rats or the homozygous rats were identified according to an electrophoresis result.
In the present disclosure, in step S2), the specific primers include primers of Ptgds-L-S, Ptgds-L-A, Ptgds-R-S, and Ptgds-R-A, with nucleotide sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.
In the present disclosure, in step S2), a reaction system of the PCR amplification includes: 2.5 μl of a template DNA at 500 ng/μl, 2.5 μl of each of the Ptgds-L-S, the Ptgds-L-A, the Ptgds-R-S, and the Ptgds-R-A that are at 10 μmol/L, 5 μl of a 10× buffer, 5 μl of dNTP at 2.5 mmol/L, 0.5 μl of Eazy-taq, and supplementing to 50 μl with water; and
the PCR amplification includes: pre-denaturation at 98° C. for 2 min; denaturation at 98° C. for 20 sec×30; annealing at 55° C. for 20 sec×30; extension at 72° C. for 10 sec×30; terminal extension at 72° C. for 5 min; and cooling at 16° C. for 2 min.
In the present disclosure, step 6) specifically includes the following steps:
6.1) the heterozygous rats selected in step 5) are used as F0-generation heterozygous rats, caged with the wild-type rats, then a gene identification is conducted on obtained offspring I, and the heterozygous rats are selected from the offspring I as F1-generation rats;
6.2) the F1-generation rats with the wild-type rats are caged, then a genetic identification is conducted on obtained offspring II, and the heterozygous rats are selected from the offspring II as F2-generation rats; and
6.3) rats generated by conducting self-breeding within the group of F2-generation rats are used as F3-generation rats, and a genetic identification is conducted to select homozygous rats as the Ptgds gene knockout rat model.
The following described a method for constructing a rat model by a specific example.
In this example, a constructed rat Ptgds gene was located on chromosome 3 of the SD rats, with only one transcript: Ptgds-201.
Therefore, the model was constructed with the Ptgds-201 transcript as an object.
As shown in
A specific sgRNA (single-guide RNA) of the Ptgds gene of rat mediated the cleavage of DNA by a Cas9 nuclease to produce specific DSBs (Double-Stranded Breaks), and an entire encoding sequence of the gene was deleted.
Referring to
In this example, two target sequences Ptgds-sgRNA1 and Ptgds-sgRNA2 were designed at a Ptgds gene locus;
In the above sequence, several genes in the box were PAM recognition sites, which were used to recognize a target sequence of a cleavage position; and a rat model was formed by injecting Ptgds-sgRNA1, Ptgds-sgRNA2 and CAS9 proteins into fertilized eggs to delete the PTGDS gene.
With a CRISPR/Cas9 system, an intron sequence fragment (15 bp, lowercase, bold and underline) associated with the Ptgds gene (SEQ ID NO:6, 2,935 bp, uppercase) and a sequence fragment (SEQ ID NO:13, 2,929 bp, uppercase, bold and underlined) between exon 1 and exon 7 of the Ptgds gene, that is, a sequence fragment with a total length of 2,944 bp was subjected to targeted knockout.
CTCCAAGCAAATGGCTGCTCTTCCAATGCTGTGGACCGGGCTGGTCCTCTTGGGTC
TCTTGGGATTTCCACAGACCCCAGCCCAGGGCCATGACACAGTGCAGCCCAACTTT
CAACAAGACAAGGTGAGAGGGTCCCCTACCCCACACCCGAGGAAACAGAAACCTC
AGGTCAGAGCCAGGCTTTCTCTCACAAGAGAGGGTGCGTTGGGCGCTGTCAGCCA
TGGGAGCTGTCTGGAACCGCGCTGGCACACAGCCTGGTTGGTCCACCTGACTCCG
CCAGGAATGTGGCTCTGATACCCACTTTACCGGAAGAGTAGACTGGGGCGAGCACT
GGGACAAAGACGGGAGCTCAACATCCTGGGGAAGGAAGGGGTCAATGAGGCAATG
AGCCAGCCTACTAGAGAGAGAGAGGGGCGTGGATGCTACCAGAACCTGTGTGTGG
GAGGAGTCAGAGTAGGGAAGGCCAGCCCACTAGGGTCTGCCCATGAGGGGCGCAT
GGTGCAGACCCGGGCATCCACTGGTCACAGTTCCTGGGGCGCTGGTACAGCGCGG
GCCTCGCCTCCAATTCAAGCTGGTTCCGGGAGAAGAAAGAGCTACTGTTTATGTGC
CAGACAGTGGTAGCTCCCTCCACAGAAGGCGGCCTCAACCTCACCTCTACCTTCCT
AAGGTGAGACAAGGGGGTGTGGCAAGTTTCGGGACAGAAGGCCCCACAACCCTGT
CTGGGGGACATCCTGGGGCTTGTTCCCTTACATCAGGGGTAATCTACCCACAGGAA
AAACCAGTGTGAGACCAAGGTGATGGTACTGCAGCCGGCAGGGGTTCCCGGACAG
TACACCTACAACAGCCCCCGTGAGTGAGCCACTTCCTTATCTGGGTAAATTCTGAG
GTAAATGCTGGCAGACTGTGCAGCCCCCTGTCCCAAAAGGTGGGGATAATGGTCAC
ACCACAAGGGTCAGTCATCCAAGACCAGACCTGATTGTGAATCTGCCTCAGGCACA
CAGGGCTACCTCTCTCCAGGGACTTTGGCCTCTCTGAAACCCAGCCACATTCTTCC
AGGCCCCTTTCCTGTCCAAATGAAATTTCCCAGTACTCTGCTGCCCAAGTGGGTCA
CATACAGGCATTCCCCAAATCCTACCCACATTTCATAGCTCCTATCCAAGTACCTCTT
TCCATGCCTCACCTGATCTATGGATTCCCACCAGAACCCTATTTCCTTGGCCTTCCT
GCTATATTGTAACTCAGCCTGATGATTTCTTGAGTCTAAGTGTTTTCTGCCCTCTCCC
CAAGATTCATGGTTTGGAGTTAGTGTTCAGGAAGGAAGCTAGAGATTGGGTGGTGG
CCACCCAGGGGAGCACAGGGAAAGAAGCCAAAGCAGGGGTGGAGGAGGAAGGCC
TGAGACCCTCCCCACAGAGAAGCCCACAAAGGCCACCCCCTCCAAGCAGAGGGAG
ATAGTGATGTGGGAGCCACATGTCTTAATCAGTGTCATTTCTTGGGTTCCCAGACTG
GGGCAGCTTCCACTCCCTCTCAGTGGTAGAAACCGACTACGATGAGTACGCGTTCC
TGTTCAGCAAGGGCACCAAGGGCCCAGGCCAGGACTTCCGCATGGCCACCCTCTA
CAGTAGGTATCCCAGCCCACAGGCCCACGCACAGGGCAGATGCCTGAGGTTGGAA
ACAGACCAAGGCCTAACCCAGAGGACAGTAACGAAGGTGTGTGGGGGCAGGGCGA
GGGCTTTTCACCTCCTGACACCGGCCCCTTCTTTATCTACCAGGCAGAGCCCAGCT
TCTGAAGGAGGAACTGAAGGAGAAATTCATCACCTTTAGCAAGGACCAGGGCCTCA
CAGAGGAGGACATTGTTTTCCTGCCCCAACCGGGTGAGGGAGGCTAAGCTGCTGA
GGAGGGAATTAGTGCAGATTAGTGCAGCCTGTGGACTGGGGAGAGTGTGGCCGCC
TACTAGTCCAGGGGCTCCAAGGAAAGAAATGGAGGTGTCAGTCTGTCCCGACAGTA
CCTCGACCTGCAGCCCCCTTTATTGGGAACCCTCTTCCTGGTGGACACCTCGCTGC
CCTGTCTGCCAGCCCCCTAGCTAGGGATTTAGGGGCACTAACAGATGGAGAAAGAC
ACCTTTTATGTTTTAAAGAACAGATTGGAGCAGGAGTGGGATGGAGTCTGAAGTGT
GGGGCTCAGCCTTGGGGAGGCTTCGTAAAGTCCAGGGAGAAGACAAAGTCCTGGT
GACTGTGGGTCTAAGCCTGATACTGACTACTTCCCTGGGCTTCTTTCTCAACAGATA
AGTGCATTCAAGAGTAAACACAGGTGAGAGAAGTCAGTCACAGGTAACACATGGTA
AGTGCCATTTACTCACTCAACATAAGACCACTGAGTGCTCATGTGACCACGGAGTG
CGGGCTGGGGTGGGGGGGATGCAGCTGCCCAAGGACTGTCCAAGTGAGACAGCCA
GAGAGAAAGGACAGTTCCAATTCCAGTGGCAGGAATAGAGCTGATGGCCAAGGGT
TCATGGGAGAAGGATAACAGCAATGGGAAGGGACCGCCCCATGAAGCCCATCCTGC
AAAATGAGTCTCCAAGGAACCAGAATGGACAAGATCGGGAAGGGACTGGTGGCCA
GGGATGGACATGGCGAGTCAGAGGGCTGGCTCCTCACCTGTGCTGTTGACTGAGA
CTCTGAGACCATAGGCCCTGGAGGGATACCCTAGGAGGCCCTGGCCGGAAGTGTT
GTTTGGGCCCCACTGGGCTCAGGGTGCTGCCCTCATCACTGATGGCTCTTGTTCTT
CTGTGCAGGTGATGTGGCCTCAGGACTCCCGTGCTCTGTCACTCTTGAGACCCAAG
CCCTGGCTCCCCAAAGACCTTCTCCGCCCTCCAGCTTTGCCTTGGTGGAGAAATAA
AATCCAAAG
CAAGTCagacctcggcttttgtctgtctgtcctccgggccatcactatagccctcttataaatttctcagtatgatgacca
aaagttggaagggcaaaaaggagtcgtttaacagatagagtgggctggagaggctgcctgtacctctgctcctaccccagccctctgaccaga
gRNA was designed through sequence alignment; since it was a large fragment deletion gene, two gRNAs were designed for each target site to be cut, but only one gRNA was used for each site, and the gRNA with a higher off-target score was preferentially used, that was, the sgRNA with a low off-target probability was selected; another gRNA was alternative, and the gene was knocked out using the alternative gRNA when the higher-scoring gRNAs failed to work.
The DNA fragment of sgRNA was amplified by PCR with a sgRNA-Vector as a template, and then recovered by gel as a template for in vitro transcription of sgRNA, and then recovered by the in vitro transcription of sgRNA and purification, and stored in a −80° C. refrigerator for later use.
The purified sgRNA and the Cas9-mRNA were injected into embryos of SD rat, and the embryos were transplanted into fallopian tubes of surrogate recipient rats to obtain neonatal rats.
Neonatal rats were obtained 21 d after embryo transfer, and genotype identification was completed about 2 weeks after birth.
Genotyping of neonatal rats was analyzed by genotype identification. Rats around 14 d after birth were numbered by toe clipping method and subjected to genotype identification.
Step 1: Extraction of genomic DNA
(1) Digestion
About a week after the rat was born, 0.5 cm of the rat toe was cut and placed in a 1.5 ml EP tube. After slight centrifugation, 500 μl and 0.5 μl of proteinase K (concentration: 20 mg/ml, dissolved in pH 7.4, 20 mmol/L Tris and 1 mmol/CaCl2, stored in a 50% glycerol buffer solution at −20° C.), were mixed well and digested in a 55° C. water bath overnight;
A formula of lysis solution included: 100 mmol/L Tris at pH 8.0, 5 mmol/L EDTA at pH 8.0, 0.5% SDS, and 1.17 g/100 ml NaCl.
1) The centrifuge tube was removed from the water bath and allowed to stand at room temperature for 10 min to 15 min, such that the sample was cooled to room temperature, the centrifuge tube was inverted to mix well, and centrifuged at 13,000 rpm at room temperature for 15 min.
2) 400 μl of a supernatant was pipetted into another new centrifuge tube. An equal volume of isopropanol was added, and the tube was turned up and down gently to mix thoroughly. At this time, a white flocculent precipitate appeared, and the tube was centrifuged at 12,000 rpm for 10 min at room temperature, and the supernatant was discarded.
3) The centrifuge tube was rinsed with 700 μl of cold 75% ethanol, and gently turned up and down to mix well. Centrifugation was conducted at 12,000 rpm for 5 min at room temperature, and all the supernatant was removed by suction.
4) The centrifuge tube was inverted on absorbent paper to blot dry the ethanol. After air-drying, the DNA was dissolved with 50 μl of sterile ddH2O at 55° C. for 2 h (if not being used immediately, the DNA was stored at −20° C.).
5) The concentration of DNA was detected, and 100 ng to 200 ng of the DNA was used as a PCR template.
Step 2: PCR amplification was conducted with specific primers using the genomic DNA as a template to obtain an amplification product; and
forward and reverse PCR primers were designed for about 200 bp to 300 bp of upstream and downstream regions of the target.
(1) The primer information was shown in Table 1.
TCCAAGCAAATGGCTGCTCTTCCAATGCTGTGGACCGGGCTGGTCCTCTTGGGTCT
CTTGGGATTTCCACAGACCCCAGCCCAGGGCCATGACACAGTGCAGCCCAACTTTC
AACAAGACAAGGTGAGAGGGTCCCCTACCCCACACCCGAGGAAACAGAAACCTCA
GGTCAGAGCCAGGCTTTCTCTCACAAGAGAGGGTGCGTTGGGCGCTGTCAGCCAT
GGGAGCTGTCTGGAACCGCGCTGGCACACAGCCTGGTTGGTCCACCTGACTCCGC
CAGGAATGTGGCTCTGATACCCACTTTACCGGAAGAGTAGACTGGGGCGAGCACT
GGGACAAAGACGGGAGCTCAACATCCTGGGGAAGGAAGGGGTCAATGAGGCAAT
GAGCCAGCCTACTAGAGAGAGAGAGGGGCGTGGATGCTACCAGAACCTGTGTGTG
GGAGGAGTCAGAGTAGGGAAGGCCAGCCCACTAGGGTCTGCCCATGAGGGGCGC
ATGGTGCAGACCCGGGCATCCACTGGTCACAGTTCCTGGGGCGCTGGTACAGCGC
GGGCCTCGCCTCCAATTCAAGCTGGTTCCGGGAGAAGAAAGAGCTACTGTTTATGT
GCCAGACAGTGGTAGCTCCCTCCACAGAAGGCGGCCTCAACCTCACCTCTACCTTC
CTAAGGTGAGACAAGGGGGTGTGGCAAGTTTCGGGACAGAAGGCCCCACAACCCT
GTCTGGGGGACATCCTGGGGCTTGTTCCCTTACATCAGGGGTAATCTACCCACAG
GAAAAACCAGTGTGAGACCAAGGTGATGGTACTGCAGCCGGCAGGGGTTCCCGGA
CAGTACACCTACAACAGCCCCCGTGAGTGAGCCACTTCCTTATCTGGGTAAATTCT
GAGGTAAATGCTGGCAGACTGTGCAGCCCCCTGTCCCAAAAGGTGGGGATAATGG
TCACACCACAAGGGTCAGTCATCCAAGACCAGACCTGATTGTGAATCTGCCTCAGG
CACACAGGGCTACCTCTCTCCAGGGACTTTGGCCTCTCTGAAACCCAGCCACATTC
TTCCAGGCCCCTTTCCTGTCCAAATGAAATTTCCCAGTACTCTGCTGCCCAAGTGG
GTCACATACAGGCATTCCCCAAATCCTACCCACATTTCATAGCTCCTATCCAAGTA
CCTCTTTCCATGCCTCACCTGATCTATGGATTCCCACCAGAACCCTATTTCCTTGGC
CTTCCTGCTATATTGTAACTCAGCCTGATGATTTCTTGAGTCTAAGTGTTTTCTGCC
CTCTCCCCAAGATTCATGGTTTGGAGTTAGTGTTCAGGAAGGAAGCTAGAGATTGG
GTGGTGGCCACCCAGGGGAGCACAGGGAAAGAAGCCAAAGCAGGGGTGGAGGAG
GAAGGCCTGAGACCCTCCCCACAGAGAAGCCCACAAAGGCCACCCCCTCCAAGCA
GAGGGAGATAGTGATGTGGGAGCCACATGTCTTAATCAGTGTCATTTCTTGGGTTC
CCAGACTGGGGCAGCTTCCACTCCCTCTCAGTGGTAGAAACCGACTACGATGAGT
ACGCGTTCCTGTTCAGCAAGGGCACCAAGGGCCCAGGCCAGGACTTCCGCATGGC
CACCCTCTACAGTAGGTATCCCAGCCCACAGGCCCACGCACAGGGCAGATGCCTG
AGGTTGGAAACAGACCAAGGCCTAACCCAGAGGACAGTAACGAAGGTGTGTGGGG
GCAGGGCGAGGGCTTTTCACCTCCTGACACCGGCCCCTTCTTTATCTACCAGGCAG
AGCCCAGCTTCTGAAGGAGGAACTGAAGGAGAAATTCATCACCTTTAGCAAGGAC
CAGGGCCTCACAGAGGAGGACATTGTTTTCCTGCCCCAACCGGGTGAGGGAGGCT
AAGCTGCTGAGGAGGGAATTAGTGCAGATTAGTGCAGCCTGTGGACTGGGGAGAG
TGTGGCCGCCTACTAGTCCAGGGGCTCCAAGGAAAGAAATGGAGGTGTCAGTCTG
TCCCGACAGTACCTCGACCTGCAGCCCCCTTTATTGGGAACCCTCTTCCTGGTGGA
CACCTCGCTGCCCTGTCTGCCAGCCCCCTAGCTAGGGATTTAGGGGCACTAACAG
ATGGAGAAAGACACCTTTTATGTTTTAAAGAACAGATTGGAGCAGGAGTGGGATG
GAGTCTGAAGTGTGGGGCTCAGCCTTGGGGAGGCTTCGTAAAGTCCAGGGAGAAG
ACAAAGTCCTGGTGACTGTGGGTCTAAGCCTGATACTGACTACTTCCCTGGGCTTC
TTTCTCAACAGATAAGTGCATTCAAGAGTAAACACAGGTGAGAGAAGTCAGTCACA
GGTAACACATGGTAAGTGCCATTTACTCACTCAACATAAGACCACTGAGTGCTCAT
GTGACCACGGAGTGCGGGCTGGGGTGGGGGGGATGCAGCTGCCCAAGGACTGTC
CAAGTGAGACAGCCAGAGAGAAAGGACAGTTCCAATTCCAGTGGCAGGAATAGAG
CTGATGGCCAAGGGTTCATGGGAGAAGGATAACAGCAATGGGAAGGGACCGCCCC
ATGAAGCCCATCCTGCAAAATGAGTCTCCAAGGAACCAGAATGGACAAGATCGGG
AAGGGACTGGTGGCCAGGGATGGACATGGCGAGTCAGAGGGCTGGCTCCTCACCT
GTGCTGTTGACTGAGACTCTGAGACCATAGGCCCTGGAGGGATACCCTAGGAGGC
CCTGGCCGGAAGTGTTGTTTGGGCCCCACTGGGCTCAGGGTGCTGCCCTCATCAC
TGATGGCTCTTGTTCTTCTGTGCAGGTGATGTGGCCTCAGGACTCCCGTGCTCTGT
CACTCTTGAGACCCAAGCCCTGGCTCCCCAAAGACCTTCTCCGCCCTCCAGCTTTG
In this example, the reaction system and reaction conditions of PCR amplification were shown in Table 2.
(3) The electrophoresis detection was conducted on the amplification product using agarose gel.
1.5 g of agarose was placed in a conical flask, added with 50 ml of a 1×TAE buffer (the TAE buffer was composed of Tris base, acetic acid, and EDTA) , and a small beaker was inverted at the bottle mouth. The mixture was boiled by heating in a microwave oven for 3 times until the agarose was completely melted, and shaken well to obtain a 3.0% agarose gel solution.
A plexiglass inner tank (gel preparation tank) in the electrophoresis tank was washed, air-dried, and a gel preparation glass plate was added. The glass plate and edges of both ends of the inner tank were sealed using scotch tape to form a mold. The inner tank was placed in a horizontal position and a comb was placed in a fixed position. The agarose gel liquid cooled to about 65° C. was mixed well and poured on the inner tank glass plate carefully, such that the gel solution was slowly spread until a uniform layer of gel was formed on the entire surface of the glass plate. The glass plate was allowed to stand at room temperature until the gel was completely solidified, the comb was slightly pulled vertically and the scotch tape was removed, then the gel and the inner tank were put into an electrophoresis tank. 1×TAE running buffer was added until the gel plate was immersed.
The DNA sample and the loading buffer were mixed on a spot plate or a parafilm, where a final dilution of the loading buffer was not less than lx. The samples were added to the sample grooves of the gel plate by a 10 μl micropipette separately. After adding a sample, the tip for sample loading was replaced to prevent contamination, and the gel surface around the sample well was not damaged when loading the samples. (Note: before loading the samples, a sequence of sample loading was recorded).
After loading the samples, the gel plate was immediately energized for electrophoresis, at a voltage of 60 V to 100 V, and the samples moved from a negative electrode (black) to a positive electrode (red). As the voltage increased, an effective separation range of the agarose gel decreased. When bromophenol blue moved to about 1 cm from a lower edge of the gel plate, the electrophoresis was terminated.
5) After electrophoresis, the gel was removed and stained with a 1×TAE solution containing 0.5 μg/ml ethidium bromide for about 20 min, and then rinsed with water for 10 min.
The gel was observed under ultraviolet light, and the presence of DNA showed a red fluorescent band, which was then photographed and stored by a gel imaging system.
Step 3: identification and discrimination of rats
The heterozygous rats or the homozygous rats were identified according to the electrophoresis result.
Determination basis for PCR amplification results: negative (WT) Ptgds+/+ showed one band: 609 bp (a sequence length between L-S and L-A); heterozygote (HZ) Ptgds+/− showed two bands of 609 bp and 786 bp (the remaining sequence length after knockout of 2,944 bp in wild-type); homozygote (HO) Ptgds−/− showed one band: 786 bp.
In this example, 8 samples (denoted as D33 to D42) were selected, and the Ptgds gene knockout rats were obtained by the above method. The electrophoresis results of PCR amplification were shown in
According to the electrophoresis results of
Rats identified as heterozygous (Ptgds+/−) were bred as F0-generation heterozygous rats.
The F0-generation rats and wild-type SD rats were caged together, and the heterozygous rats identified from offspring were used as F1-generation rats; the F1-generation rats were continued to be caged with the wild-type SD rats, and the heterozygous rats identified from offspring were used as F2-generation rats; and rats produced by self-breding within the group of the F2-generation rats were used as F3-generation rats, and homozygous rats (Ptgds−/−) among them were used as the Ptgds gene knockout rat model. In reproduction of each generation, the phenotype of the Ptgds knockout rats in offspring was determined by the above-mentioned genetic identification method.
Breeding passages of F1-F3 lasted 16 months. There were 2 heterozygous rats (2 females) and 8 wild-type rats (3 females, 5 males) produced in the F0 generation; 2 heterozygous rats (1 female, 1 male) and 9 wild-type rats (4 females, 5 males) generation produced in the F1 generation; 5 heterozygous rats (3 females, 2 males) and 5 wild-type rats (2 females, 3 males) produced in the F2 generation; 33 offspring mice, including 4 homozygotes (2 females, 2 males, with a homozygous rate of about 12.12%), 19 heterozygotes (12 females, 7 males), and 9 wild-type mice (5 females, 4 males) bred in the F3 generation. During the breeding, 2 adult mice of F3 generation died, showing a mortality rate of about 6.06%. In the F3 generation, Ptgds wild-type (−/−, 9 rats): heterozygous type (+/−, 19 rats): homozygous type (+/+, 4 rats) had a ratio of approximately 2.25:4.75:1.
Perimenopausal kidney yin deficiency is a syndrome manifested by deficiency of kidney yin, lack of nourishment, and internal heat with yin deficiency. The main clinical manifestations are dizziness, tinnitus, insomnia and dreaminess, dysphoria in chestpalms-soles, waist and knee pains, hot flashes, and night sweats. A large number of experimental studies have shown that perimenopausal kidney yin deficiency is related to the dysfunction of the hypothalamic-pituitary-gonadal axis, mainly manifested as abnormal body weight, increased hot flash index, decreased renal function, abnormal blood glucose, lipid metabolism disorders, and disturbance of endocrine hormone levels (estrogen, thyroid hormone, and adrenal cortex hormone) related to the hypothalamic-pituitary-gonadal axis.
Therefore, the related indicators of “hypothalamus-pituitary-gonadal axis” in Ptgds knockout rats with different phenotypes were determined; and the results were subjected to one-way statistical analysis of variance by SPSS software, and p<0.05 indicated that there was a significant statistical difference between the groups.
In perimenopausal women, due to the rapid depletion of estrogen in the body, may have abnormal weight gain caused by the disorder of fat metabolism. In perimenopausal patients with kidney yin deficiency, may also have typical degenerative changes in their internal organs. Therefore, in this study, body weight and organ index of rats were used as external indicators for the preliminary evaluation of perimenopausal fat metabolism disorder and organ degenerative changes in gene knockout rats.
Since the life span of this type of rats is shorter than that of wild-type rats (12-14 months old), three phenotypes of 8-month-old Ptgds knockout nulliparous female rats (middle-aged, negative, heterozygous and homozygous types) were anesthetized with a 2% sodium pentobarbital solution. The brain, uterus, kidney and spleen of rats were isolated, rinsed with normal saline, dried with filter papers, weighed, and the organ index was calculated (organ index %=organ weight/body weight×100%).
The results were shown in
Referring to
One week after bilateral oophorectomy of rats, a blood flow of the tail increased and showed a transient surge, and a skin temperature of the tail was proportional to the blood flow of the tail, and typical symptoms of perimenopausal hot flashes appeared. Therefore, the severity of hot flashes and the effectiveness of drugs in the treatment of hot flash symptoms were evaluated by measuring the rat tail temperature in real time.
The rat was immobilized in a immobilizer, and a tip of the rat tail was immobilized on a surface of a measuring table; a skin temperature at about 2 cm from the base of the tail of the rat within 6 h was measured with an infrared thermal imager. Data were recorded with a temperature logger, and sampled every 5 min. An average temperature of the first 15 min was used as a baseline value. After the rat was in a stable state in the immobilizer, the temperature value was recorded and a temperature change was evaluated (there were 15 data points, 6 evaluation points, a laboratory temperature was 25° C.±2° C., a measurement time was at 9:00 to 12:00).
The results were shown in
As can be seen from
Since kidney yin deficiency is related to the hyperfunction of the hypothalamus-pituitary-gonadal axis, the functions of the gonad-related organs (kidney, pancreas, thyroid and uterus) were firstly measured.
Rat serum samples were collected, and blood biochemical indicators were determined enzymatically to evaluate the kidneys, including: albumin (ALB), uric acid (UA), urea (UREA), blood glucose (GLU), total cholesterol (TC), triglyceride (TG), and creatinine (CREA).
The secretion function indexes of kidney, pancreas, thyroid and uterine glands were determined by an enzyme-linked immunosorbent assay (ELISA), including: kidney, uterine ERβ, serum adrenocorticotropic hormone (ACTH), corticosterone (CORT), insulin (INS), cyclic adenosine monophosphate (cAMP), and thyroid stimulating hormone (TSH).
The measurement results were shown in
The results of blood biochemical tests showed that the heterozygous and homozygous rats had abnormal renal function and blood lipid indexes compared with the wild-type rats, indicating that the two types of rats might have kidney damage and lipid metabolism disorders. The abnormal increase of renal function Na+-K+-ATPase, ACTH, CORT, immune cAMP and thyroid TSH further indicated that, the heterozygous and homozygous rats showed typical symptoms of kidney yin deficiency and abnormal renal function and hyperfunction of endocrine glands. Compared with wild-type rats, the decrease of ERβ level in uterus of heterozygous and homozygous rats indicated that the ovarian secretion function of rats was degenerated; this result was consistent with the decreased uterine organ index, indicating that Ptgds knockout rats could simulate the perimenopausal uterine and ovarian decline in rats. The abnormal increase of insulin levels in heterozygous and homozygous rats reflected the abnormal increase of blood glucose and the lipid metabolism disorder caused by insulin resistance in perimenopause.
Perimenopause patients with kidney yin deficiency may show typical symptoms of central degenerative diseases such as lapse of memory and senile dementia. Therefore, the memory and learning ability of Ptgds knockout rats were evaluated by a Morris water maze method. Experiments were conducted in a Morris water maze system, including a white plastic pool (130 cm in diameter, 50 cm in height, with a built-in PVC cylindrical escape platform), an automatic camera, and image tracking processing software.
Before the test, a 5-day positioning navigation training was conducted. That is, each rat (stained with yellow dye on a top of the head) was placed in titanium powder-dyed white water (23° C.±2° C.) with its head facing the pool wall at random quadrant points, and timing started at the moment of release; the timing was stopped when the rat touched the escape platform and stayed there for at least 3 sec. When finding the platform, the rats stayed on the platform for 10 sec; if the rat could not find the platform within 90 sec, the rats were guided to the platform and acclimated for 10 sec. After the formal experiment began, the original escape platform was removed; the second quadrant was used as a fixed water entry quadrant, and the escape latency, swimming distance, and times of crossing the platform of the rats were recorded within a specified time, to evaluate the learning and memory ability of the rats. The results were shown in
Referring to
In summary, the present disclosure is based on use of the CRISPR/Cas9 technology in targeted knockout of Ptgds gene in rats, thereby constructing a Ptgds −/− rat model to produce symptoms of the spontaneous kidney yin deficiency. Through breeding, PCR identification, and pathological index detection, the model can be further applied to pharmacodynamic evaluation. The model can provide a reliable and stable genetic engineering model for the study of perimenopausal symptoms of kidney yin deficiency, and lay a foundation for exploration of perimenopausal syndrome mechanism and evaluation of related drug treatment effects.
The foregoing are merely descriptions of the specific embodiments of the present disclosure, and the claimed scope of the present disclosure is not limited thereto. Any modification, equivalent replacement, improvement, etc. made within the technical scope of the present disclosure by those skilled in the art according to the spirit and principle of the present disclosure shall fall within the claimed scope of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202210191036.7 | Feb 2022 | CN | national |
