Zebrafish as a disease model for studying human hepatocellular carcinoma

Table 1 Advantages and disadvantages of constitutive and inducible expression systems

Expression systems	Advantages	Disadvantages
Constitutive	Well established, commercially available; in vitro and in vivo, successful methodology for expression of transgene	Expression of oncogenes may cause advanced/highly aggressive tumors and early lethality
Heat-shock	Expression of transgene can be induced on a single cell level	Adverse effects that may arise from the heat shock
Cre-loxP	Well established; commercially available; in vivo, successful methodology for expression of transgene	Not all tissue specific promoters are perfectly specific; leaky gene expression; two plasmid system
GAL4/UAS	Well established; in vivo, successful methodology for expression of transgene	In vivo expression of GAL4 can have side effects, probably related to immune and stress responses; two plasmid system
Mifepristone	Well established; in vivo, successful methodology for expression of transgene	Opening and closing of the switch is slow (hours to days); cell permeability of the RU-486 can be restricted
Tet-on/off-inducible	Well established; commercially available; in vitro and in vivo, successful methodology for expression of transgene	Opening and closing of the switch is slow (hours to days); cell permeability of the doxycycline can be restricted; two plasmid system

Table 2 Characteristics of three genome editing systems

Nucleases	ZFN	TALEN	CRISPR/Cas
DNA binding domain	Multiple zinc finger peptides	Transcription-activator like effectors	CRISPR-derived RNA/Single-guide RNA
Endonuclease	Fok1	Fok1	Cas9
Binding specificity of each repeat	3 bp	2 bp	1 bp
Target site length	18 to 36 bp	30 to 40 bp	23 bp
Off-target	High probability	Low probability	Variable
Libraries generation	No	Feasible, depend on technology	Yes, cloning 20 bp, oligos targeting each gene into a plasmid

Table 3 Zebrafish animal models of liver disease and hepatocellular carcinoma

Transgene name	Expression system	Liver pathology	Ref.
cnr1 (Zebrafish)	Tet-off-inducible	Steatosis	[132]
edn1 (Zebrafish)	Constitutive	Steatosis, bile duct dilation, hyperplasia and HCC	[69]
gankyrin (Zebrafish)	Constitutive	Atrophy, hypoplasia and steatosis	[123]
HBx (Human)	Constitutive	Hypoplasia and steatosis	[120]
HBx + AFB1 (Human)	Constitutive	Hepatitis, steatosis and hyperplasia	[134]
HBx + HCV (Human)	Tet-off-inducible	Intrahepatic cholangiocarcinoma	[81]
HBx + p53M214 (Human)	Constitutive	Chronic inflammation, steatosis, bile duct dilation, dysplasia and HCC	[70]
HBx + src (Human/Zebrafish)	Constitutive	Chronic inflammation, steatosis, bile duct dilation, dysplasia and HCC	[70]
HCV (Human)	Constitutive	Steatosis	[29]
HCV + TAA (Human)	Constitutive	Steatosis and HCC	[29]
kras-G12V (Zebrafish)	Mifepristone	Hyperplasia and HCC	[82]
kras-G12V (Zebrafish)	Constitutive	Hyperplasia and hepatocellular adenoma	[82]
kras-G12V (Zebrafish)	Tet-on-inducible	Hyperplasia, hepatocellular adenoma and HCC	[137]
kras-G12V + p53M214 (Zebrafish)	Constitutive	Hyperplasia and hepatocellular adenoma	[82]
kras-G12V + RhoA (Zebrafish)	Tet-on-inducible	Hyperplasia, hepatocellular adenoma and HCC	[137]
kras-G12V + RhoAG14V (Zebrafish)	Tet-on-inducible	Hyperplasia, hepatocellular adenoma and HCC	[137]
kras-G12V + RhoAT19N (Zebrafish)	Tet-on-inducible	HCC	[137]
Lc3 (Rat)	Constitutive	Investigation of liver autophagy	[141]
mdm2 (Zebrafish)	Constitutive	Atrophy, contraction and hypoplasia	[121]
MYC (Mouse)	Tet-on-inducible	Hyperplasia and hepatocellular adenoma	[83]
myca (Zebrafish)	Mifepristone	Small, typical, hypervascular and ascites of liver tumor	[143]
myca + p53M214 (Zebrafish)	Mifepristone	Small, typical, hypervascular and ascites of liver tumor	[143]
mycb (Zebrafish)	Mifepristone	Small, typical, hypervascular and ascites of liver tumor	[143]
orf A (Human)	GAL4/UAS	Delayed onset of liver tumor	[79]
src (Zebrafish)	Constitutive	Chronic inflammation, steatosis, bile duct dilation, hyperplasia, dysplasia and HCC	[70]
src + p53M214 (Zebrafish)	Constitutive	Steatosis, hyperplasia, dysplasia and HCC	[70]
UHRF1 (Human)	Constitutive	Atypical cells, dysplastic foci and HCC	[124]
UHRF1 + p53M214 (Human)	Constitutive	Atypical cells, dysplastic foci and HCC	[124]
xmrk (Xiphophorus)	Tet-on-inducible	Hyperplasia, hepatocellular adenoma and HCC	[80]
yy1 (Zebrafish)	Constitutive	Steatosis	[131]
zfBLP1 (Zebrafish)	Constitutive	Hyperplasia	[133]
zfMcl-1α (Zebrafish)	Constitutive	Hyperplasia	[133]

HCC: Hepatocellular carcinoma; HBx: Hepatitis B virus X protein; HCV: Hepatitis C virus.