Method for producing transgenic sheep by injecting lentivirus into perivitelline space

Abstract

The invention provides a method for producing transgenic sheep by injecting lentivirus into perivitelline space; lentiviral vector and virus packaging plasmid are cotransfected into 293T cells and then centrifugated after 48 hours to prepare the concrete of the lentiviral; the concrete of the lentiviral is injected into oocyte perivitelline space which is 24 to 25 hours mature, is integrated with sperms to implement in vitro fertilization, is put into culture fluid after 12 hours to be cultured, and the cleavage rate and the transgenic rate are calculated after 48 hours. Step 1: an oocyte is washed by in vitro maturation fluid for 3 to 4 times, and the 75 to 78um l / drop in vitro maturation fluid which is dripped by 25 to 30m / drop is cultured in 5 percent CO2 by 95 percent; step 2: a cumulus cell is removed from the in vitro matured oocytes, the lentiviral is injected into the cell perivitelline space and is washed by IVF washing liquid for 2 to 4 times, and fertilization fluid is dripped by 25 to 30m / drop; semen is unfrozen, the fertilization fluid is moved in, supernatant fluid is centrifugurated and removed, the sperms are precipitated and added into fertilization fluid drips and incubated; eggs which are fertilized for 12 to 18 hours are treated in the above step and then put into a four-hole culture plate to be cultured; and step 3: medicine lentiviral, egg pumping liquid, the maturation fluid, micromanipulation liquid, SOF, the fertilization fluid, the culture fluid and sheep serum are prepared.

Description

technical field [0001] The invention relates to the production of transgenic sheep, in particular to a method for producing transgenic sheep by injecting lentivirus in the perivitelline space, which can effectively improve the survival rate of embryos and the transgenic rate. Background technique [0002] Animals obtained through transgenic technology are called transgenic animals. Lentivirus (LV) is a viral member of the Retroviridae family. Traditional transgenic methods include retrovirus infection, embryonic stem cell, sperm carrier, germ cell transfection, nuclear transfer, and pronuclear microinjection, but these methods all have low transgenic rates and high costs. Defects. Because compared with the traditional pronuclear microinjection technique, perivitelline space injection does not need to insert the injection needle into the nucleus, and is not limited by the difference in the size of the pronucleus and the clarity of the nuclear membrane in different species o...

AI Technical Summary

Problems solved by technology

The above-mentioned existing methods are used in the production of transgenic cattle and pigs, but there are few reports on the production of transgenic sheep

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