All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania. The derivation of the FoxA1loxP/loxP;FoxA2 loxP/loxP;FoxA3−/− as well as HNF4αloxP/loxP mice has been reported previously (Kaestner et al. 1998 (link); Sund et al. 2000 (link); Parviz et al. 2002 (link); Gao et al. 2008 (link)). Male mice at ages 8–12 wk were jugular vein-injected with 1011 particles of AAV expressing either Cre recombinase (Addgene 107787-AAV8) or GFP (Addgene 105535-AAV8) under the control of the hepatocyte-specific thyroid-binding globulin (Tbg) promoter. Mice were genotyped by PCR of tail DNA as described in the above-mentioned papers. Validation of HNF4α depletion was shown by Armour et al. (2017) (link).
107787 aav8
Manufactured by Addgene
107787-AAV8 is an adeno-associated virus (AAV) vector. AAV vectors are commonly used for gene delivery applications in biological research. This specific vector, 107787-AAV8, contains the AAV8 capsid serotype, which is a commonly used capsid for in vivo gene delivery experiments.
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Lab products found in correlation
Lenti x 293t, by Takara Bio (1 mentions)
Fugene hd, by Promega (1 mentions)
Albumin cre mice, by Jackson ImmunoResearch (1 mentions)
D12492, by Research Diets (1 mentions)
Flp deleter mice, by Jackson ImmunoResearch (1 mentions)
Anti nk1.1 clone pk136, by BioXCell (1 mentions)
C57bl 6j mice, by Jackson ImmunoResearch (1 mentions)
4 protocols using 107787 aav8
1
Hepatocyte-specific HNF4α Depletion
2
Lentivirus and Retrovirus Production
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To produce lentiviruses and retroviruses, 6-well plates of 70% confluent Lenti-X 293T(Clontech) cells were transfected with 1.5 μg of transfer vector, 0.5 μg pMD2.G and 1.0 μg of psPAX2 for lentivirus or 1.0 μg of gag/pol for retrovirus using Fugene HD (Promega) according to the manufacturer’s instructions. Supernatant was collected after 48 h and frozen at –80 °C. AAV packaging for LDTS-eLIR and LDTS-LIR mut were performed with AAV8 serotype at the University of Pennsylvania Vector Core (#V6014S; 8.462e13 GC/ml, #V6016S; 1.069e14 GC/ml, respectively). AAV to express Cre in the liver was purchased from Addgene (#107787-AAV8).
3
Liver-Specific Slc25a47 Knockout Mouse Model
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All the animal experiments in this study were performed in compliance with protocols approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center. The Slc25a47 floxed (Slc25a47flox/flox) mouse was generated by in vitro fertilization of homozygous sperm (UC David) from Slc25a47tm1a (EUCOMM)Hmgu targeting exons 5 and 6 of the Slc25a47 gene in C57BL/6J background. A floxed LacZ-neomycin cassette on the Tm1a allele was removed using a flippase (FLP)/Frt deletion by breeding Slc25a47flox/flox with FLP deleter mice (Jackson Laboratory, Stock No. 009086). Slc25a47flox/flox mice were bred with Albumin Cre mice (Jackson Laboratory, Stock No. 003574) to generate liver-specific Slc25a47 deletion mice (Slc25a47Alb-Cre). Mice were kept under a 12-h:12-h light–dark cycle at ambient temperature (22 to 23 °C) and had free access to food and water. Mice were maintained on a regular chow diet or fed with a high-fat diet (60% fat, D12492, Research Diets) starting from 6 wk of age for 6 wk. All mice were fasted for 6 h before killing. To acutely deplete Slc25a47, we injected 7-wk-old Slc25a47flox/flox mice with 1.5 × 1011 genome copies of AAV8-TBG-Cre (Addgene, 107787-AAV8) or AAV8-TBG-null (control, Addgene, 105536-AAV8) through tail vein injection.
4
In Vivo Genetic Perturbation Screening
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C57BL/6J mice (strain 000664) and LSL-Cas9 mice (strain 026175) were purchased from the Jackson Laboratory. Mice were either singly-or group-housed with a 12-hour light-dark cycle (light from 7 AM to 7 PM, dark from 7 PM to 7 AM) in a specificpathogen-free animal facility with unlimited access to food and water. To deliver lentivirus, up to 100 µL of lentivirus in PBS was injected into the temporal vein of postnatal day one mice. For protein depletion tests, mice were injected with 1.25 x 10 7 transduction units (TU) of sgRNA-mCherry lentivirus. For the screen, mice were injected with 5 x 10 7 TU of sgRNA-mCherry lentiviral library. For validating Cxorf38 and Tap1, mice were injected with 1 x 10 7 TU of an equal mixture of sgAAVS1-mTurq2 and sgCxorf38-mCherry or sgTap1-mCherry lentiviruses. To deliver AAV-Cre, a stock solution of AAV8-TBG-Cre (Addgene 107787-AAV8) was diluted in PBS to a total volume of 20 µL and injected intraperitoneally into postnatal day five mice. For protein depletion tests, the screen, and validating hits, mice were injected with 2 x 10 11 GC of AAV-TBG-Cre. To deplete NK cells, 15 µg/g of anti-NK1.1 (clone PK136, BioXCell) was injected intraperitoneally every three days beginning at postnatal day five. All animal procedures were approved by the Massachusetts Institute of Technology Committee on Animal Care.
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