tetO-ALN | Addgene 43918 product information

This webpage contains legacy information. The product is either no longer available from the supplier or has been delisted at Labome.

product summary

company name :

Addgene

product type :

cDNA

product name :

tetO-ALN

catalog :

43918

citations: 2

Reference
Powell S, O Shea C, Townsley K, Prytkova I, Dobrindt K, Elahi R, et al. Induction of dopaminergic neurons for neuronal subtype-specific modeling of psychiatric disease risk. Mol Psychiatry. 2021;: pubmed publisher
Addis R, Hsu F, Wright R, Dichter M, Coulter D, Gearhart J. Efficient conversion of astrocytes to functional midbrain dopaminergic neurons using a single polycistronic vector. PLoS ONE. 2011;6:e28719 pubmed publisher

product information

Catalog Number :

43918

Product Name :

tetO-ALN

article :

doi	10.1371/journal.pone.0028719
id	6382
pubmed_id	22174877

bacterial resistance :

Ampicillin

cloning :

backbone

FU-tetO-Gateway

backbone_mutation

backbone_origin

Addgene plasmid 43914

backbone_size

8600

promoter

sequencing_primer_3

sequencing_primer_5

vector_types

Mammalian Expression

Lentiviral

Other

Doxycycline inducible

growth notes :

CTCTGCCAAG; ASCL1-REV: CATCTCCTGCTTGCTTTAACAGAGAGAAGTTCGTGGCACC GGATCCGAACCAGTTGGTGAAGTC; LMX1B-FWD: CTCTCTGTTAAAGCAAGCAGGAGATGTTGAAGAAAACCCC GGGCCTATGTTGGACGGCATCAAG; LMX1B-REV: ACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCTCCGG ATCCGGAGGCGAAGTAGGAACT; NURR1-FWD: GAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGG CCCTATGCCTTGTGTTCAGGCG; B2-NURR1-REV: GGGGaccactttgtacaagaaagctgggt[A]GAAAGGTAAAGTGTCCAG (extra [A] added to maintain reading frame with C-terminal V5-tag). The 3 ORFs were then joined via recombinant PCR such that viral 2A peptide sequences separate the three genes, using B1-ASCL1-FWD and B2-NURR1-REV. A glycine-serine-glycine (GSG) linker was included upstream of each 2A sequence to facilitate protein cleavage. The ASCL1-P2A-LMX1B-T2A-NURR1(ALN) cassette was cloned into pDONR221, and subsequently cloned into FU-tetO-Gateway via Gateway recombination to produce the tetO-ALN vector. Cleavage at 2A sites was verified by performing in vitro transcription and translation using the TnT T7 Coupled Reticulocyte Lysate System (Promega L4610) in the presence of biotinylated lysine (Promega L5061). 5 L of each TnT-generated protein sample was resolved via PAGE, transferred to PVDF membrane, incubated with streptavidin-conjugated horseradish peroxidase (Cell Signaling Technology 3999, 1:2000), and detected with ECL Plus (GE Healthcare RPN2132).
GGGGacaagtttgtacaaaaaagcaggctACCATGGAAA
G
To make a drug-inducible lentiviral vector that is compatible with Gateway recombination (Invitrogen), the depositing laboratory removed the OCT4 open reading frame from FU-tetO-hOCT4 (Addgene plasmid 19778) and replaced it with a Gateway cassette cloned from pEF-DEST51 (Invitrogen) to generate FU-tetO-Gateway (Addgene plasmid #43914). Open reading frames of human ASCL1, LMX1B, and NURR1 were purchased from Open Biosystems and amplified using the following PCR primers: B1-ASCL1-FWD:

growth temp :

Use recA cells, such as STBL3 (Invitrogen)

origin :

pi :

alt_names

ASCL1

LMX1B

NURR1 (NR4A2)

cloning

clone_method	Gateway Cloning
cloning_site_3
cloning_site_5
promoter	TRE promoter, Tet-ON
sequencing_primer_3	WPRE-R
sequencing_primer_5	LNCX; T7
site_3_destroyed
site_5_destroyed

entrez_gene

aliases	ASH1, HASH1, MASH1, bHLHa46
gene	ASCL1
id	429

aliases	FSGS10, LMX1.2, NPS1
gene	LMX1B
id	4010

aliases	HZF-3, IDLDP, NOT, NURR1, RNR1, TINUR
gene	NR4A2
id	4929

genbank_ids

DQ894571/EL736297/EU832740 (Genbank Accession Numbers)

100009031/13957/100067769 (Open Biosystems IDs)

mutation

Gylcine-serine-glycine (GSG) linker followed by viral 2A peptide sequence inserted between each ORF

name

ASCL1-2A-LMX1B-2A-NURR1

shRNA_sequence

size

3800

species

9606

Homo sapiens