Plasmid plusmega kit

Mosquito larvae were collected in Goundry village, Burkina Faso (latitude 12.5166876, longitude −1.3921092) using described methods^{39 (link)}, reared to adults, and were typed for species by the SINE200 X6.1 assay^{40 (link)}. DNA from 60 A. coluzzii were pooled at equal volume and sheared using an S220 ultrasonicator (Covaris) to produce DNA fragments 800–1000 bp in length. Subsequently, DNA was processed as described for the STARR-seq assay^{30 (link)}, cloned into the plasmid pSTARR-seq_fly (AddGene 71499), transformed into MegaX DH10B T1R Electrocomp Cells (Invitrogen), cultured in LB+ ampicillin (1 ug/ml), and plasmid DNA was purified using the Plasmid Plus Mega Kit (Qiagen). The Anopheles gambiae PEST AgamP4 genome assembly available at Vectorbase was used as the reference genome (https://www.vectorbase.org/organisms/anopheles-gambiae/pest/agamp4).

To generate the pT3-EF1a-ChREBP-IRES-Luciferase vector (ChREBP-luc), the pT3-EF1A-MYC-IRES-luc was used as a donor vector. The cloning of ChREBP, with N-terminal HA tag, into the donor vector was performed by In-Fusion cloning (Takara Bio, 638910). The pT3-EF1A-MYC-IRES-luc plasmid was a gift from Amaia Lujambio (Addgene plasmid # 129775). The CMV-SB13 transposase plasmid was kindly provided by Dr. Scott Lowe (MSKCC, New York). TERT, c-myc and Jarid1B were also stably overexpressed in the liver of C57BL6/J mice using the SB transposon system. These oncogenes were overexpressed using the respective plasmids: pT3-EF1A-mTert (gift from Amaia Lujambio (Addgene plasmid # 162555)), PT3-EF1a-c-myc (gift from Xin Chen (Addgene plasmid # 92046)). To generate the pT3-EF1a-Jarid1b vector, the pT3TS vector was used as a donor vector (gift from Stephen Ekker (Addgene plasmid # 31830)). For these 3 oncogenes, the SB13 transposase was stably overexpressed using the PT2/C-Luc//PGK-SB13 plasmid which was a gift from John Ohlfest (Addgene plasmid # 20207). All constructs were verified by nucleotide sequencing and vector integrity was confirmed by restriction enzyme digestion. Vectors for hydrodynamic delivery were produced using the QIAGEN plasmid PlusMega kit (QIAGEN, 12981). Equivalent DNA concentration between different batches of DNA was confirmed to ensure reproducibility among experiments.

A sterile 0.9% NaCl solution/plasmid mix was prepared containing DNA. We prepared 11.4 μg of pT3-EF1a-MYC-IRES-luciferase (MYCluc), 10 μg of px330-sg-p53 (sg-p53), and a 4:1 ratio of transposon to SB13 transposase-encoding plasmid dissolved in 2 mL of 0.9% NaCl solution and injected 10% of the weight of each mouse in volume as previously described^{72 (link)}. Because two independent “hits” are required for tumor formation in C57BL/6 mice^{97 (link)}, only those hepatocytes that receive the three plasmids (transposon-based, transposase, and CRISPR-based) will have the potential to form tumors. Mice were injected with the 0.9% NaCl solution/ plasmid mix into the lateral tail vein with a total volume corresponding to 10% of body weight in 5 to 7 s. Vectors for hydrodynamic delivery were produced using the QIAGEN plasmid PlusMega kit (QIAGEN). Equivalent DNA concentration between different batches of DNA was confirmed to ensure reproducibility among experiments.