ERalpha36 knock-down PC12 cell strain and establishing method thereof

Abstract

The invention discloses an establishing method of an ERalpha36 knock-down PC12 cell strain. An shRNA (short hairpin ribonucleic acid) technology is used, an ERalpha36-shRNA transfected PC12 cell is utilized to establish the ERalpha36 knock-down PC12 cell strain. Immunocyte fluorescence, immunocytochemistry and Western blot technologies are utilized to detect the ERalpha36 in the cell strain, and the fact proves that the cell strain is successfully established. A growth curve and doubling time of the cell are determined. The establishment of the ERalpha36 knock-down PC12 cell strain provides a powerful experimental material for researching functions of the ERalpha36 in the central nervous system.

Description

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an ERα36 knockdown PC12 cell line and a construction method thereof. Background technique

[0002] Estrogen (E) has a wide range of biological activities in living organisms, not only regulating many target tissues such as reproductive tract, breast, bone and cardiovascular system, but also acting on neural circuits related to cognitive function, Participate in the regulation of neurotransmitters such as serotonin, dopamine, glutamate, GABA and acetylcholine and the activities of various neuropeptides, and affect synaptic plasticity and learning and memory. At the same time, estrogen also plays an important role in many processes such as the differentiation and development of nerve cells, the induction of neuron survival and axon growth, and the formation of new neurons. Alzheimer's disease (AD) is a neurodegenerative disease that seriously threatens the ...

Examples

Embodiment 1

[0049] Example 1. Acquisition of plasmid ERα36-shRNA

[0050] Plasmid ERα36-shRNA was donated by Dr. Wang Zhaoyi, Creighton University Medical Research Center, USA. It was cloned by DNA oligonucleotide sequences: SEQ ID NO: 1 and SEQ ID NO: 2 genes, and recombined into pRNAT-U6 .1 / Neo vector to construct specific ERα36-shRNA.

[0051] (1) Synthesis of shRNA sequence and DNA template: The shRNA DNA template corresponding to ERα36 gene is: SEQ ID NO: 1, SEQ ID NO: 2 and the artificially synthesized sequences.

[0052] (2) Construction of ERα36-shRNA expression vector

[0053] Schematic diagram of the construction of ERα36-shRNA expression vector, such as figure 1 . Take 1ug / ul each of the two single-stranded DNA templates of SEQ ID NO: 1 and SEQ ID NO: 2, mix well, heat at 95°C for 10min, let stand at room temperature for 1h, dilute to a final concentration of 10ng / ul for use, and use T 4 DNA ligase was ligated with the linearized vector pRNAT-U6.1 / Neo digested with BamH I a...

Embodiment 2

[0065] Example 2. Transfection of plasmid ERα36-shRNA into PC12 cells

[0066] (1) Before transfection:

[0067] Cells were seeded in a 6-well plate at a concentration of 6×10 5 PC12 cells / mL, 2mL per well, placed in 5% CO at 37°C 2 Cultivate in an incubator, and after 24 hours, the cell adhesion concentration reaches 90% for transfection;

[0068] (2) Transfection process: For the transfection method, refer to the instructions of Lipofectamine 2000 (Invitrogen, USA).

[0069] (a) Add 20 μL of Lipofectmine 2000 to 250 μL of Opti-MEM, mix gently, and let stand at room temperature for 5 minutes;

[0070] (b) Gently mix 2 μL of ERα36-shRNA plasmid with a concentration of 2 μ / μL and 250 μL of Opti-MEM;

[0071] (c) Mix the mixture obtained in steps (a) and (b), and let stand at room temperature for 20 minutes;

[0072] (d) Replace the medium in the 6-well plate of step (1) with RPMI1640 medium without serum and antibiotics, 2 mL per well;

[0073] (e) Add 500 μL of the mixed...

Embodiment 3

[0076] Example 3. Screening of positive cell lines

[0077] (1) Add G418 with a final concentration of 500 μg / mL to each well of the 6-well plate in step (f) to screen positive cell lines, and replace G418 with a final concentration of 500 μg / mL and 15% newborn bovine serum every 3 to 5 days ( NCS) RPMI1640 selection medium at 37°C in 5% CO 2 Cultivate in the incubator for 10-14 days;

[0078] (2) Pick a single clone and continue to store at 37°C in 5% CO 2 Cultivate in an incubator for 3 to 4 weeks; name the positive cell line obtained as PC12-36L;

[0079] (3) Add G418 to the RPMI1640 medium of 15% NCS at a final concentration of 250 μg / mL, at 37°C in 5% CO 2 Maintain screening in the incubator, and replace the above medium every 3 to 5 days;

technical field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an ERα36 knockdown PC12 cell line and a construction method thereof. Background technique

[0002] Estrogen (E) has a wide range of biological activities in living organisms, not only regulating many target tissues such as reproductive tract, breast, bone and cardiovascular system, but also acting on neural circuits related to cognitive function, Participate in the regulation of neurotransmitters such as serotonin, dopamine, glutamate, GABA and acetylcholine and the activities of various neuropeptides, and affect synaptic plasticity and learning and memory. At the same time, estrogen also plays an important role in many processes such as the differentiation and development of nerve cells, the induction of neuron survival and axon growth, and the formation of new neurons. Alzheimer's disease (AD) is a neurodegenerative disease that seriously threatens the ...

Examples

Embodiment 1

[0049] Example 1. Acquisition of plasmid ERα36-shRNA

[0050] Plasmid ERα36-shRNA was donated by Dr. Wang Zhaoyi, Creighton University Medical Research Center, USA. It was cloned by DNA oligonucleotide sequences: SEQ ID NO: 1 and SEQ ID NO: 2 genes, and recombined into pRNAT-U6 .1 / Neo vector to construct specific ERα36-shRNA.

[0051] (1) Synthesis of shRNA sequence and DNA template: The shRNA DNA template corresponding to ERα36 gene is: SEQ ID NO: 1, SEQ ID NO: 2 and the artificially synthesized sequences.

[0052] (2) Construction of ERα36-shRNA expression vector

[0053] Schematic diagram of the construction of ERα36-shRNA expression vector, such as figure 1 . Take 1ug / ul each of the two single-stranded DNA templates of SEQ ID NO: 1 and SEQ ID NO: 2, mix well, heat at 95°C for 10min, let stand at room temperature for 1h, dilute to a final concentration of 10ng / ul for use, and use T 4 DNA ligase was ligated with the linearized vector pRNAT-U6.1 / Neo digested with BamH I a...

Embodiment 2

[0065] Example 2. Transfection of plasmid ERα36-shRNA into PC12 cells

[0066] (1) Before transfection:

[0067] Cells were seeded in a 6-well plate at a concentration of 6×10 5 PC12 cells / mL, 2mL per well, placed in 5% CO at 37°C 2 Cultivate in an incubator, and after 24 hours, the cell adhesion concentration reaches 90% for transfection;

[0068] (2) Transfection process: For the transfection method, refer to the instructions of Lipofectamine 2000 (Invitrogen, USA).

[0069] (a) Add 20 μL of Lipofectmine 2000 to 250 μL of Opti-MEM, mix gently, and let stand at room temperature for 5 minutes;

[0070] (b) Gently mix 2 μL of ERα36-shRNA plasmid with a concentration of 2 μ / μL and 250 μL of Opti-MEM;

[0071] (c) Mix the mixture obtained in steps (a) and (b), and let stand at room temperature for 20 minutes;

[0072] (d) Replace the medium in the 6-well plate of step (1) with RPMI1640 medium without serum and antibiotics, 2 mL per well;

[0073] (e) Add 500 μL of the mixed...

Embodiment 3

[0076] Example 3. Screening of positive cell lines

[0077] (1) Add G418 with a final concentration of 500 μg / mL to each well of the 6-well plate in step (f) to screen positive cell lines, and replace G418 with a final concentration of 500 μg / mL and 15% newborn bovine serum every 3 to 5 days ( NCS) RPMI1640 selection medium at 37°C in 5% CO 2 Cultivate in the incubator for 10-14 days;

[0078] (2) Pick a single clone and continue to store at 37°C in 5% CO 2 Cultivate in an incubator for 3 to 4 weeks; name the positive cell line obtained as PC12-36L;

[0079] (3) Add G418 to the RPMI1640 medium of 15% NCS at a final concentration of 250 μg / mL, at 37°C in 5% CO 2 Maintain screening in the incubator, and replace the above medium every 3 to 5 days;

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