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Conditional Cas9 expression induced swine trophoblastic cell line and establishment method and application thereof

A technology of trophoblast cells and establishment methods, applied in the field of porcine trophoblast cell lines that conditionally induce Cas9 expression and its establishment, can solve the problems of negative effects on fetal growth and development, and achieve the effect of simple operation and convenient use

Inactive Publication Date: 2016-06-01
AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the embryonic stage of animals, when the expression of the target gene is not required, the addition of Dox will have a negative impact on the growth and development of the fetus

Method used

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  • Conditional Cas9 expression induced swine trophoblastic cell line and establishment method and application thereof
  • Conditional Cas9 expression induced swine trophoblastic cell line and establishment method and application thereof
  • Conditional Cas9 expression induced swine trophoblastic cell line and establishment method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0032] Example 1 A method for establishing a porcine trophoblast cell line that conditionally induces Cas9 expression

[0033] see figure 1 , for the present invention to establish the road map of the porcine trophoblast cell line that conditionally induces the expression of Cas9, the method for establishing the porcine trophoblast cell line that conditionally induces Cas9 expression comprises the following specific steps:

[0034] (1) Combining the Tet-on inducible expression system with CRISPR-Cas9 technology to obtain a conditionally expressed Cas9 gene, which is then packaged with lentivirus, concentrated and purified to obtain a titer of 10 8 TU / mL lentivirus solution;

[0035] (2), add 100 μ L of step (1) to the complete medium (DMEM medium containing 10% FBS, 55 μM β-mercaptoethanol, 1% L-glutamine and 1% Penicilin-Stretomycin) added with 6 μg / mL polybrene lentiviral solution with 10 4 A characteristic porcine trophoblast cell line (TSCs isolated from the ectoderm of...

Embodiment 2 Embodiment 1

[0041] Example 2 Detection of CAS9 protein activity of the porcine trophoblast cell line of Example 1

[0042] After the HU6 promoter (266bp) + target sequence + SgRNA (85bp) fragment is transferred into the porcine trophoblast cell line, a 105bp sgRNA sequence can be expressed, of which 20bp is a specific target sequence, which can guide the targeted editing of the genome by the CAS9 protein (like image 3 A), wherein the front part of the N part of the sequence is the HU6 promoter sequence, the N part of the sequence is the designed target gene sequence, the back is the SgRNA hairpin structure sequence, and the last 6 Ts are the terminator structure, which is the sequence structure of the sgRNA;

[0043] Using specific primers and overlapping PCR technology can obtain HU6+Sohlh1 gene target sequence (20bp)+SgRNA sequence DNA fragment (such as image 3 B); when Dox was added to induce, the cellular genome Sohlh1 was cleaved and destroyed (eg image 3 C); when Dox was not ad...

Embodiment 3

[0044] Example 3 Expression of AQP1, AQP3, AQP5 and AQP9 in Sow Placenta at Different Stages of Pregnancy

[0045] RT-PCR results showed that the expression of AQP1, AQP3, AQP5 and AQP9mRNA was detected in the placenta of sows on the 25th and 60th day of gestation (such as Figure 4 ).

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Abstract

The invention discloses a conditional Cas9 expression induced swine trophoblastic cell line and an establishment method and application thereof. The method includes: subjecting a lentiviral vector, for controlling Cas9 gene expression, of a Tet-on system to virus packaging, concentrating and purifying; incubating lentivirus with a typical swine trophoblastic cell line, changing fluid, adding puromycin for drug screening, diluting cells to single ones, and culturing to obtain the single-cell-source swine trophoblastic cell line with Cas9 expression under Tet-on regulation. The swine trophoblastic cell line is simple in operation and convenient to use and has a potential utilization value in researching of key functional genes of some trophoblast cells and screening of swine gene target spots. The cell line is expected to be a significant cell material for researching swine placenta development and applicable to correlation researches on swine early-stage placenta development and uterine pregnancy mechanisms and also has a high reference value in research of human early embryonic development.

Description

technical field [0001] The invention belongs to the technical field of cytology, and more specifically, the invention relates to a porcine trophoblast cell line that conditionally induces the expression of Cas9 and its establishment method and application. Background technique [0002] Trophoblastic stem cells (TSCs) and embryonic stem cells (embryonic stem cells, ESCs) are two types of stem cells established from early mammalian blastocysts, but the two stem cells have great differences. TSCs are derived from the trophoblast cells of blastocysts, located in The periphery of the blastocyst can only be differentiated into placental cells in vivo / in vitro; while ESCs are derived from the inner cell mass cells of the blastocyst, which can develop into individual fetuses in vivo, and can infinitely proliferate and differentiate into multiple cells of any type in vitro. functional stem cells. [0003] In humans, rats, and rabbits, ESCs can be induced to differentiate into TSCs b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N5/10
CPCC12N15/86C12N5/0605C12N2740/15043
Inventor 朱翠白银山陈庄蒋宗勇
Owner AGRO BIOLOGICAL GENE RES CENT GUANGDONG ACADEMY OF AGRI SCI
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