PAMAM-D mediated sperm vector method for cultivating transgenic mammalian

A mammalian and transgenic technology, applied in the field of genetic engineering, can solve problems such as poor repeatability and large randomness of gene transfer

Inactive Publication Date: 2008-10-01
天津市泌尿外科研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the shortcomings of large randomness and poor repeatability of gene transfer in the existing sperm carrier transgenic technology, the purpose of the present invention is to use the structure and performance characteristics of PAMAM-D to establish a PAMAM-D-mediated method for preparing transgenic mammals. Sperm carrier method to improve gene transfer efficiency and enhance the stability of gene transfer effect

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Example 1: Exogenous DNA configuration and dosage: Plasmid DNA must be linearly integrated on the chromosome, and the gene configuration that enters the cell is circular when it integrates with the chromosome, the probability of damage is high, the integration rate is low, and it affects other cells. Therefore, before forming a complex with PAMAM-D, the exogenous DNA is digested rationally and becomes a linear configuration with cohesive ends. Too small a dose will affect the transfection effect, but a large dose of DNA will inhibit sperm motility and activate endogenous nucleases in sperm cells to cause degradation. Therefore, it is necessary to determine the amount of exogenous DNA according to the type of sperm and the size of exogenous DNA to be prepared for transgenic animals. The transgenic animal to be prepared in this example is a pig, and the exogenous DNA is a hDAF gene recombinant plasmid, which is cut into a linear line with the endonuclease Sac I, and the e...

Embodiment 2

[0017]Example 2: Screening of PAMAM-D synthesis algebra and dose: The efficiency of PAMAM-D-mediated exogenous DNA transfer varies with different cell types, and the ability to enter the nucleus may also vary with different cell types. In the complete PAMAM-D, only the synthesis generation > G5 can effectively mediate the transfer of exogenous DNA, and the transfer rate increases exponentially with the increase of the synthesis generation within a certain range. The charge density of PAMAM-D molecules is decisive in the process of complex formation and combination with sperm. With the increase of the charge ratio between PAMAM-D and exogenous DNA (such as > 20), the low-density, soluble complex produce more. When the concentration of PAMAM-D increases, its charge ratio to DNA increases, leading to increased complex formation. The transfection rate is also related to the size of the exogenous DNA molecule. The larger the nucleotide molecule, the lower the concentration of PAMA...

Embodiment 3

[0018] Embodiment 3: the preparation of PAMAM-D / exogenous DNA complex: with the PAMAM-D of different synthetic algebras and doses of embodiment 2, in the SFM solution that the concentration of PAMAM-D is 1g / L, add the result of embodiment 1 Alternative different amounts of exogenous DNA to form a series of PAMAM-D / exogenous charge ratios of 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1 The DNA complex is used to transfect sperm, and it is detected by in situ hybridization method. The optimal complex with high transfection rate and no damage to sperm viability is determined by the amount of exogenous DNA and the number and dosage of PAMAM-D synthesis. good combination conditions. This embodiment is aimed at 1×10 7 / ml of porcine spermatozoa, the compound was prepared with 4 μg of hDAF linear plasmid and PAMAM-D (G7) with a charge ratio of 20:1, which met the above optimal combination conditions. The PAMAM-D / exogenous DNA complex prepared under the optimal combination conditions wil...

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Abstract

The present invention provides a method of culturing PAMAM-D mediated sperm vector of transgenic mammalian, including following steps: foreign gene enzyme is cut into a linear configuration of end cohesive, determining its dosage range aimed at different sperms; screening PAMAM-D in the composition algebra which is more than G5 and ranges of exogenous charge ratio 2-50:1, transfecting sperm by the exogenous DNA has high effect, and also keeping the optimum combination condition of the exogenous DNA usage of sperm activity and PAMAM-D generation and dose, so as to prepare PAMAM-D/exogenous DNA compound, adequately washing collected sperm under the monitor of the supernatant fluid IF-1 after washed; transfecting sperm outside of compound by incubates method, the female animal is conceived by individually selecting single sperm of transfection exogenous DNA, or sperm of transfection exogenous DNA formed by injecting in testicle, in vitro fertilization embryo transfer, in vivo artificial insemination and natural mating, cultivating transgenic animal.

Description

technical field [0001] The present invention belongs to genetic engineering. Background technique [0002] At present, there is an important problem to be solved urgently in the preparation of transgenic animals, that is, the microinjection method of fertilized eggs commonly used is expensive equipment, complicated operation technology, low efficiency, and high production cost. People are trying to find a simple and efficient transgenic method , the sperm carrier method has received great attention. [0003] In 1971, Brackett et al used isotope-labeled SV40 virus DNA to co-incubate with rabbit sperm, and confirmed for the first time that sperm can be used as a carrier for exogenous gene transfer. Eighteen years later, after Arezzo (1989) fertilized eggs with starfish sperm adsorbed to pSRVCAT or pSV2CAT, exogenous genes were expressed in embryos. In the same year, Lavitrano et al. (1989) used mouse epididymis sperm as an exogenous DNA carrier, and after in vitro fertilizat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C12N15/87A01K67/027
Inventor 王广有杨惠祥乔宝民刘春雨李胜芝张玥马腾骧
Owner 天津市泌尿外科研究所
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