Page 91 - GPD-4-1

Gene & Protein in Disease Oral-ERT in PD knockout mice with tobrhGAA

dose (containing either 25 or 75 μg of tobrhGAA per 2 μg/mL and 1 to 25 μL of sample. Samples were read at
dose) mixed 3:1 with apple juice as a safe, non-invasive 595 nm absorbance within 60 min.
oral administration technique and measured the vertical
99
hang-time, as previously described by Raben et al. 109 2.13. Glycogen content assay
In a 96-well microtiter plate, 25 μL of sample, 25 μL of 0.05
2.9. Oral-ERT in GAA KO mice
M sodium phosphate pH 6.5, and 0.5 μg of amyloglucosidase
GAA KO mice aged approximately 5 months were fed (Sigma #A-1602) were added and incubated overnight at
either 50 mg or 150 mg of tobrhGAA ground seeds 37°C. Glycogen standards (rabbit liver, 2 mg/mL) and
112
daily mixed with peanut butter in Petri dishes (12/12-h D-glucose standards were measured at 400, 200, and 100 μM.
light/dark cycle) (B6,129-Gaa tm1Rabn /J mice). Tissues, Thereafter, 20 μL of Eton Bioscience glucose assay solution
109
urine, blood, weight, and serum were collected. Mice (#1200031002) was added and incubated for 30 min at 37°C.
were tested every 3 weeks for motor activity using a The reaction was stopped with the addition of 25 μL of 0.5 M
running wheel (RW-Mini-Mitter Co., Inc., OR, USA); acetic acid and read at 490 nm absorbance. 112
forelimb muscle strength using a GSM (Columbus
Inst.); motor coordination and balance using a Rotarod 2.14. Ames mutagenicity test
(AJATPA, Expert Industries, India); open-field mobility Escherichia coli (E. coli) DH5a and DH5a/pUC19 cells
using a 5-min video to determine distance traveled; were plated on NZ media with ampicillin and various
and spontaneous alternative learning using a T-maze amounts of tobrhGAA lysate, and the colony-forming
(Stoelting, IL, USA). units (CFUs) were counted. 113-117
2.10. Determination of antibodies against tobrhGAA 2.15. Complete blood count differentials
using ELISA
Complete blood count (CBC) differentials were performed
In Falcon plates #3912, 1 μg/well of Sephadex G100 purified on blood smear slides using Giemsa–Wright staining.
human placental GAA 111 was added in 100 μL PBS and
incubated overnight at 4°C. This was then washed 3 times 2.16. Extraction of DNA and RNA from seeds for NGS
with PBS and blocked with 200 μL of PBS with 1% bovine and RNA transcriptome profiling
serum albumin (BSA) and 0.05% Tween20 for at least DNA and total RNA were extracted from wild-type (Nicotiana
60 min at room temperature. Thereafter, 1 μL of serum in tobacum L. cv. xanthi) and tobrhGAA seeds using commercial
100 µL of PBS was added and incubated overnight at 4°C. kits (Thermo Scientific GeneJET Plant RNA Purification Kit-
This was washed 3 times with PBS, once with PBS and K0801; GeneJET Plant DNA Purification Kit-K0791). The
0.05% Tween20, and twice with PBS. Then 100 μL of goat NGS genome sequences and RNA expression profiles were
anti-mouse IgG-horse radish peroxidase (BioRad #172- performed at BGI.com, and global proteomic profiling was
1101) was added (diluted 1:500 in PBS/BSA/Tween20) performed using ultra-performance liquid chromatography-
and incubated overnight at 4°C. This was washed 3 times mass spectrometry (UPLC-MS/MS) at bioproximity.com.
with PBS, once with PBS and 0.05% Tween-20, and twice
with PBS. The cells were stained with 100 μL of 3,3',5,5’– 2.17. Nicotine levels in leaves and seeds
tetramethylbenzidine (Sigma #T5525) in 9 mL of 0.05 The levels of nicotine in tobrhGAA#3 seeds and leaves
M phosphate-citrate buffer, pH 5.0 (Sigma #P4922) with were measured by gas chromatography-mass spectroscopy
20 μL of 30% H O for 30 – 60 min at room temperature. (GC/MS) at Avogado Analytical (LLC, Salem, NH, USA).
2
2
Reactions are stopped with the addition of 50 μL 1 N HCl
and read at 450 nm absorption. 2.18. Statistical analysis
2.11. Endotoxin determination The Student’s t-test (1-tail and 2-equal variance) for
probability associated with a population and standard
Endotoxin in various amounts of tobrhGAA seed lysate was deviation (SD) based upon the population were determined
determined using the Pierce LAL Chromogenic Endotoxin using Microsoft Excel software, and results were considered
Quantitation Kit (#88282) according to the manufacturer’s significant at P ≤ 0.05.
instructions. 111
3. Results
2.12. Bradford protein assay
3.1. Nicotine levels in leaves and seeds
For the protein assay, 150 µL of reagent (BioRad #500-
0006) diluted 1:5 with water in a 96-well microtiter plate. The nicotine levels were measured using GC/MS. The
Standards were serial dilutions of BSA from 1,000 to tobrhGAA seeds or leaves contained <5 ng of nicotine per

Volume 4 Issue 1 (2025) 5 doi: 10.36922/gpd.1760