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Lin28a

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LIN28A is a protein that regulates the expression of certain genes involved in cellular development and differentiation. It plays a role in the regulation of let-7 microRNA family members, which are important for controlling cell fate and stemness. LIN28A is expressed in embryonic stem cells and various types of cancer cells.

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Lab products found in correlation

Iwr 1, by Selleck Chemicals (2 mentions) Glis1, by Addgene (2 mentions) Pmxs oct4, by Addgene (2 mentions) Nanog, by Addgene (2 mentions) Epz004777 idot1l, by Aobious (1 mentions) Mircury lna mirna inhibitor, by Qiagen (1 mentions) Tdtomato c1 vector, by Addgene (1 mentions) Nebuilder hifi dna assembly technology, by New England Biolabs (1 mentions) Lin28b, by Addgene (1 mentions)

4 protocols using lin28a

1

Generation of Polycistronic Reprogramming Vectors

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The DOT1L inhibitor EPZ004777 (iDOT1L) was purchased from AOBIOUS Inc. (Gloucester, MA, USA). WNT inhibitor IWR1 was purchased from Selleckchem (Houston, TX, USA). The constructs pMXs-OCT4, NANOG, LIN28A and GLIS1 were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KLF4, MYC and SOX2 (KMS) was described in our previous study (Wang et al., 2017 (link)). To clone the pMXs-GNL or NL polycistronic vector, the coding sequences for human NANOG, LIN28A and GLIS1 were PCR-amplified from the above-mentioned Addgene constructs. The amplified DNA sequences for each gene were then inserted into linearized pMXs vectors (Cell Biolabs, San Diego, CA, USA) using an In-Fusion kit (Clontech Inc., Mountain View, CA, USA). 2A sequences (Carey et al., 2009 (link); Ryan and Drew, 1994 (link); Ryan et al., 1991 (link)) were inserted between each gene.
Wang L., Su Y., Huang C., Yin Y., Chu A., Knupp A, & Tang Y. (2019). NANOG and LIN28 dramatically improve human cell reprogramming by modulating LIN41 and canonical WNT activities. Biology Open, 8(12), bio047225.
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2

Cloning of SunTag-FL2 Construct

Cited 1 time
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For cloning of the SunTag-FL2 construct, we used the SINAPs plasmid from the Singer lab (Addgene #84561)44 and a tdTomato-FL2 (containing the 3’UTR of FL2) plasmid previously cloned in-house using the tdTomato-C1 vector (Addgene #54653), a human FL2 clone in pANT7_cGST (DNASU, Arizona State University, Tempe, AZ, clone: HsCD00403041) and a human FL2 3’UTR construct (Switchgear Genomics #S811553). The Ubc promoter, flag tag and SunTag reporter were cloned from the SINAPs construct to replace the CMV promoter and tdTomato reporter of the tdTomato-FL2 plasmid using NEBuilder HiFi DNA Assembly technology (NEB).
The plasmids used for IMP RBP overexpression, GFP-ZBP133 (link), GFP-IGF2BP2 and GFP-IGF2BP2 KH3 mutant39 (link), were from the Singer laboratory. The Lin28 plasmids were purchased from Addgene; Lin28A (#51371), Lin28B (#51373) and Lin28A-mCCHC (#51372). The miRNA let-7 inhibitor sequence (3’-CUCCAUCAUCCAACAU-5’) was previously validated for broad let-7 family inhibition (Frost & Olson, 2011). The custom miRCURY LNA miRNA inhibitor was purchased from Qiagen (Cat num 339146).
Birnbaum R., Biswas J., Singer R.H, & Sharp D.J. (2023). mRNA Localization and Local Translation of the Microtubule Severing Enzyme, Fidgetin-Like 2, in Polarization, Migration and Outgrowth. bioRxiv.
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3

Reprogramming B Cells to Induced Pluripotency

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B cells were reprogrammed as described by Barrett et al. (2014) (link). Briefly, 1 × 106 B cells were nucleofected using B-Cell Nucleofector Kit (VPA-1001; Lonza) with 4 plasmids containing the following genes: SOX2, OCT4, KLF4, LIN28A, MYCL, SV40LT, and shTP53 (Addgene).
Waldhorn I., Turetsky T., Steiner D., Gil Y., Benyamini H., Gropp M, & Reubinoff B.E. (2022). Modeling sex differences in humans using isogenic induced pluripotent stem cells. Stem Cell Reports, 17(12), 2732-2744.
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4

Reprogramming Factors for iPSC Generation

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The DOT1L inhibitor EPZ004777 was purchased from AOBIOUS Inc (Gloucester, MA, USA). Wnt inhibitor IWR1 was purchased from Selleckchem.com (Houston, TX, USA). The constructs pMXs-OCT4, NANOG, LIN28A, GLIS1, and FOXH1 were purchased from Addgene (Cambridge, MA, USA). Construction of the polycistronic vector pMXs-KMS was described in our previous study27 (link). Retroviral shRNA construct specific for FOXH17 (link) were purchased from Addgene.
Wang L., Su Y., Huang C., Yin Y., Zhu J., Knupp A., Chu A, & Tang Y. (2019). FOXH1 Is Regulated by NANOG and LIN28 for Early-stage Reprogramming. Scientific Reports, 9, 16443.
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