B-Tcf21-iCreERT2 mice

Basic Information

Description

This Tcf-iCre/ERT2 model is an efficient tool to study various gene functions when crossed with mice with different loxP site-flanked genes of interest, especially in studies of natural mesenchyme-derived tissue.

Targeting strategy

Details

 

 Genotyping PCR Primer

Primer	Sequence (5’-3’)	Tm (℃)	Product Size (bp)
Primer-WT-F	GACTGCCAGATCCCACCTCAAAC	62	WT:415bp
Primer-WT-R	GGAGAAGGCCTTGCTCAGCAC	63
Primer-WT-F	GACTGCCAGATCCCACCTCAAAC	62	Mut:265bp
Primer–Mut-R	GCACACAGACAGGAGCATCTTC	59

 PCR Reaction System

Reaction Component	Volume(µL)	Final Concentration
ddH2O	14	–
10×Taq buffer	2	1×
10 mM dNTPs	0.5	250 mM each
10 mM Primer-F	0.5	0.25 mM
10 mM Primer-R	0.5	0.25 mM
2.5 U/mL Taq DNA Polymerase Polymerase	0.5	0.0625 U/mL
100-200   ng/mL Template DNA	2	10-20 ng/mL

PCR Steps

Step	Temp.	Time	Cycles
1	95℃	5 min	1
2	95 ℃	30 s	30
3	61 ℃	30 s
4	72 ℃	1 kb / min
5	72 ℃	10 min	1
6	4℃	hold	1

Determination of Genotypes

Identification of Genotypes According to PCR Results

Primer-WT-F/Primer-WT-R		Primer-WT-F /Primer-Mut-R	Genotype
Mutation	Wild Type	Mutation
N*	N	Y	Mut/Mut
N*	Y	Y	Mut/+
N	Y	N	+/+

N*: PCR product was too long to detect Mut/Mut: representing homozygous
N: No expected PCR product detected Mut/+: hemizygous
Y: Expected PCR product detected. +/+: wild type allele

Agarose Gel Electrophoresis

Gene Editing Results

Number	Strain Number	Genotype
1	1SY2-009	Mut/ +
2	1SY2-011	Mut/ +
3	1SY2-012	Mut/ +
4	1SY2-014	Mut/ +
5	1SY2-015	Mut/ +
6	1SY2-019	Mut/+
7	1SY2-020	Mut/ +
8	1SY2-022	Mut/ +
9	1SY2-026	Mut/ +
10	1SY2-031	Mut/ +
11	1SY2-032	Mut/ +

 

References

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