Methods for enhancing genome engineering efficiency
A genome and engineering technology, applied in genetic engineering, chemical instruments and methods, biochemical equipment and methods, etc., can solve the problems of less modified cells and obstacles to the routine implementation of transient gene editing
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Embodiment 1
[0173] Example 1: Co-delivery of TSA with a construct containing a tdTomato reporter gene (i.e. pLH-Pat5077399-70Subi-tDt) by microprojectile bombardment in maize immature embryos without trichostatin A (TSA) pretreatment Improved instantaneous conversion efficiency.
[0174] Procedure: Preparation of maize immature embryos for bombardment: 8-10 days after pollination, corn ears with immature embryos 0.8 to 1.8 mm in size (ie A188 or Hi II) were harvested. Spikes are sterilized with 70% ethanol for 10-15 min. After brief drying in the laminarhood, the upper -1 / 3 of the kernels were removed from the ears with a shark scalpel and the immature embryos were carefully removed from the kernels with a spatula. Freshly isolated embryos were plated onto bombardment target areas in osmotic culture plates (see below) with the embryonic leaves facing up. Prior to bombardment, plates were wrapped in parafilm and incubated at 25°C in the dark for 4-20 hours.
[0175] For use with 100μg g...
Embodiment 2
[0185] Example 2: TSA bombarded with tdTomato reporter construct pGEP359 ( Figure 4 ) co-delivery boosted transformation efficiency
[0186] Type II callus induction and selection: As described in Example 1, Hi II immature embryos with a size of 0.8-1.8 mm were isolated and immediately placed on the type II callus induction culture with the cotyledon face up. On a basis (see below), the density was 10-15 embryos per plate (100 mm in diameter). The plates were wrapped with parafilm and the embryos were cultured in the plates at 27°C in the dark until type II callus appeared (~2 weeks). Loose Type II calli were picked under a stereoscope and transferred to Type II callus selection medium (see below). Repeat this process 2-3 times and discard the embryos 4 weeks after induction. The pre-embryonic stage of Type II callus was carefully selected under a stereoscope based on looseness (highly loose type), morphology (no embryo-like structure), color (fresh, white, translucent). ...
Embodiment 3
[0193] Example 3: Co-delivery of trichostatin A (TSA) by microprojectile bombardment with construct pLH-Pat5077399-70Subi-tDt improves transient transformation of sugar beet loose callus.
[0194] Sugar beet callus induction: Young leaves from sugar beet sprouts cultured in vitro in sprout medium (see below) were cut into small pieces (squares, 3-5 mm in size) in a laminar flow hood and placed in On medium (see below) at a density of 10-15 pieces per plate (100 mm diameter), adaxial side up. The plates were wrapped with parafilm and the leaf segments were cultured on the plates for 6-8 weeks at 23°C in the dark until callus appeared.
[0195] Preparation of sugar beet calli for bombardment: Loose type fresh calli were harvested under a stereoscope and transferred to the bombardment target area (see below) in sugar beet infiltration medium (single layer, no overlap). Prior to bombardment, plates were wrapped with parafilm and incubated at 25°C in the dark for 4-20 hours.
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