Technical Serial No. 2: Application of Cre-loxP site-specific recombination system in the study of conditioned target gene function in mice

Author: Yu Xiaofeng, Chief Scientist of Saiye Biology/Vice President of China

Highlights of the article

Text continued

6.

1.

Even if the targeted knock-in strategy is used to construct a Cre mouse model, it may affect the expression of the corresponding genes, leading to a certain pathological change phenotype in the mouse model
.
In particular, a construction strategy of knocking Cre into the 5'end of an endogenous gene is adopted
.
For example, Foxg1-Cre knock-in/knockout mice means that when Cre knocks in the endogenous Foxg1 gene locus, the gene is also knocked out.
The Foxg1 gene is involved in the development of the mouse brain hemisphere.
Therefore, the Cre homozygous is small.
The rat died during the perinatal period
.
Although Foxg1-Cre heterozygous mice can still perform specific targeting effects on brain tissues, and achieve specific knockout of target genes in mouse brain hemispheres, preoptic/optic vesicles, olfactory epithelial nerves, and pharyngeal vesicles.
Purpose
.

2.
Toxic effects of high expression of Cre recombinase

The large accumulation of Cre recombinase in cells can directly cause DNA damage and cell death
.
Studies have shown that for transgenic mice that express Cre highly, their survival and reproductive ability is reduced
.
CAG-Cre and CD2-Cre mouse models are typical examples
.

Myh6-Cre mouse is a commonly used mouse model specific to myocardium, constructed with the help of aMyHC promoter and random insertion of transgene strategy
.
Comparing the effects of different expression levels of Cre in mice, it is found that non-specific recombination occurs in Cre high-expressing mice, which affects the normal function of mouse myocardium
.
However, transgenic mice with low Cre expression have no non-specific recombination effect
.
Researchers believe that highly expressed Cre and certain so-called secret loxP (cloxP) sites in mice have non-specific recombination and cleavage effects, which can disrupt the function of related genes
.

The high expression of Cre in neuronal cells can cause damage to mouse brain development
.
Research and analysis of Nestin-Cre and two Nestin-CreERT2 transgenic mouse strains confirmed that all homozygous Nestin-Cre mice have delayed embryonic development and decreased neuronal proliferation, increased aneuploidy, and apoptosis.
Developmental defect phenotypes such as cerebellar disease and hydrocephalus in mice
.

High levels of Cre expression can also directly affect the function of retinal pigment epithelial cells
.
After comparing and analyzing different retinal pigment epithelial cells (RPE) specific Cre transgenic mice, it is found that commonly used Trp1-Cre transgenic mice themselves are accompanied by disordered RPE monolayer cell structure, RPE area atrophy, loss of retinal photoreceptor cell function, and small glue Pathological changes such as plasma cell activation
.

RIP (Ins2)-Cre mice , which are commonly used as metabolic diseases , have also seen glucose tolerance reactions or induced diabetes, which may be related to impaired insulin secretion
.

Reports have shown that induced intestinal-specific Villin-CreERT2 mice, activated by TAM, can use the secret loxP site to cause non-specific DNA damage
.

3.
Cre reproductive system reorganization and the effect of mouse sex

Through the study of 64 different nervous system-related Cre mouse models, it is found that 64.
1% of the mice have reproductive system reorganization, and 29 species (82.
8%) have gender differences, of which the male parent Cre mice have complete or selective reproduction Sex recombination activity accounted for 62.
1%, while female Cre mice accounted for only 20.
1%
.
Only 17.
2% of Cre mice have similar germline reorganization effects
.
For example, the offspring of Foxg1-Cre/floxed double-gene edited male mice and wild female mice will have a certain degree of gene knockout (68.
8%), but no reproductive cells of Foxg1-Cre female mice Role
.

Analyze the possible causes and influencing factors of the germline recombination of these Cre mice, including the mouse model construction strategy itself , such as transgene or targeted knock-in strategy, promoter selection, Cre insertion site and expression level, 5 The'end or 3'end knock-in, IRES and 2A connection method can all be potential influencing factors
.
For example, the so-called endothelial cell-specific Tek-Cre (Tie2-Cre) transgenic mouse model is mostly constructed with the aid of the 2.
1 kb promoter of the Tek gene and the 10 kb intron fragment region
.
The results showed that the Cre expression activity appeared in mouse embryos at 7.
5 days and showed a certain degree of recombination activity of hematopoietic stem cell lines
.
And the female parent mice showed obvious reproductive system reorganization
.
It has been confirmed that the Tek gene regulatory sequence has an active role in the embryonic development process and germline cells, which causes the target gene to be knocked out in the germline
.

4.
Mosaicism by Cre  

Due to the changed or inconsistent Cre expression, the activity and efficiency of Cre recombination cleavage in different cells or tissues in the same mouse are different, which causes the effect of knocking out target genes in different cells or tissues, showing inconsistent phenomena
.
For example, in the Vav1-Cre or Fabp4-Cre mouse strains, the phenotypes related to the genetic changes caused by the Cre mice are also different in the offspring of the Cre mice
.
In addition, as a commonly used tool for complete knockout of target genes in a wide range of cells and tissues , EIIa-Cre mice have also been confirmed in practical applications.
Male Ella-Cre mice have obvious mosaic expression characteristics, while female Ella-Cre mice are recombined.
The cutting activity is more uniform and consistent
.
Therefore, it is recommended to use female Ella-Cre mice to mate with floxed male mice to obtain a more ideal and complete recombination effect
.

5.
Influence of mouse genetic background

Studies have found that the same Cre mouse model with different genetic backgrounds can have different recombination cleavage activities
.
For example, the abnormal eye phenotype caused by the eye-specific Le-Cre transgenic mice is related to the influence of its genetic background
.
When this Cre mouse is commonly used to mate with FVB/N strains and CBA/Ca genetic background mice for 7 generations, some Le-Cre mice of CBA/Ca strains have obvious eye abnormal phenotypes
.
Moreover, the severity and frequency of the eye phenotype of CBA/Ca strain mice can also be reversed and eliminated by backcrossing FVB mice for 2 generations
.

6.
Related issues in TAM application

CreERT2-loxP site-specific inducible recombination system requires the participation of the inducer TAM
.
However, in practical applications, there are many factors that can affect its effects, and may cause non-specific and related toxic side effects, which should be understood and paid attention to
.

(1) About the optimal dose and approach for effective induction of TAM

It is difficult to find the best TAM induction effectiveness in the application research analysis of the reported CreERT mouse strains.
Part of the reason is that different studies have applied different experimental strategies and operating procedures, including a variety of different TAMs.
preparation and dose, and other various routes of administration
.
For example, a questionnaire survey conducted by the University of Columbia to related researchers showed that the preparation method of TAM (corn oil is mostly formulated, followed by sunflower oil and peanut oil, etc.
), dosage (20mg～175 mg, etc.
), administration route and frequency (abdominal cavity) The most injections include single, 2-4, and 5 injections, followed by oral gavage, etc.
), and the induction effect detection methods (RT-PCR and immunofluorescence staining) are significantly different
.

Although most of the current studies have adopted TAM intraperitoneal injection (IP) administration , some studies have compared the effects of IP administration and oral gavage (PO) administration, and found that PO may reduce the toxicity of TAM.
More advantages
.
And it is found that elderly CreERT mice often require higher doses and longer time for TAM induction to obtain satisfactory CreERT activity
.

Regarding the optimal TAM effective induction dose and route of administration, the current relevant knowledge is still very limited, and it needs to continue to explore and accumulate
.
It is also recommended that researchers screen out the best dosage and route for the research plan based on actual conditions
.

(2) TAM induced toxicity

It is now clear that high-dose intraperitoneal administration of TAM has long-term toxic effects and can increase morbidity and mortality in mice
.
For example, extensively inducible R26CreERT2 mice, if induced with high concentrations of TAM (175 mg/kg, oral cavity for 5 consecutive days), the mice will experience thymic atrophy, severe anemia, and abnormal chromosomal rearrangement of hematopoietic cells and other hematopoietic system-related toxic reactions
.

Studies have also found that after transgenic mice expressing MerCreMer (MCM) specifically induced by myocardium , ~60-80% of MCM mice appear after medium and high doses of TAM (60～90 ug/g, abdominal cavity for 3～5 days).
Cardiac fibrosis, accompanied by increased expression of pro-inflammatory cytokines, eventually leads to heart failure and death of mice
.
However, reducing the dose or frequency of TAM induction (for example, 30 ug/g, intraperitoneal administration for 3 consecutive days) can not only maximize the specific recombination effect of Cre (~84%), but also minimize the effect of its cardiotoxicity
.
At the same time, it is recommended to use TAM-treated MCM mice as experimental controls
.

Further analysis of the MCM mouse CreERT transgene insertion site confirmed that the MCM transgene was inserted into the C1 region of chromosome 19 of the mouse genome, resulting in the deletion of an approximately ~20 kb genomic fragment containing A1cf gene exon 1 and part of intron 1 Area
.

Regarding the possible causes of TAM-induced cardiotoxicity in MCM mice , related studies believe that TAM-induced cardiac fibrosis and heart failure are not caused by the random insertion site of MCM and the level of TAM dosage itself, but because of medium and high doses of TAM.
Induction, the expression of Cre is correspondingly increased, and the possible Cre is related to the non-specific recombination cleavage of cloxP hidden sites
.
Because, (1) MCM mice of different ages have no cardiac fibrosis; (2) the same high-dose TAM induces wild B6 mice, but no mouse cardiac fibrosis changes; (3) high-dose TAM can cause MCM The increased programmed death of cardiomyocytes in mice may be related to DNA destruction and activation and decreased stability of p53 gene; (4) In vitro primary cells confirmed that high expression of Cre recombinase can directly cause programmed cell death
.

Studies have compared and analyzed two inducible CD4-CreERT2 mouse strains constructed with different strategies (random insertion and targeted knock-in).
By mating them with homozygous Rosa26-loxP-stop-loxP-YFP reporter mice, After TAM induction, observe the dynamic response of T follicular helper cells (Tfh) in vivo, and immunize mice with incomplete protein-coupled NP-KLH antigen to induce the differentiation of helper Tfh cells
.
The results showed that random insertion of high-expressing CD4-CreERT2 into transgenic mice can reduce the number of helper Tfh cells and interfere with cell survival
.
The targeted knock-in mice with low expression of CreERT2 can avoid cell loss caused by overexpression of Cre, and can still effectively exert its lymphocyte-specific recombination efficiency
.

The research reports on the toxicity induced by TAM described above are mostly related to the use of high-dose TAM and the effect of high expression of Cre
.
However, recent studies have shown that low-dose TAM (20 mg/kg) induction can also affect bone formation in mice
.
The researchers used different doses of TAM (0, 20, 40, and 200 mg/kg, abdominal cavity for 4 consecutive days) to induce 4-week-old wild B6 mice.
The results showed that induction of 20 mg/kg dose of TAM can induce femoral volume in mice.
The ratio (bone volume/total volume) increased by 153%
.
Therefore, it is suggested that even if a very low dose of TAM is used for induction, in the actual CreERT bone-specific target gene knockout/expression study, it is necessary to consider that TAM induction may affect the formation of trabecular bone and cortical bone in mice
.
( Unfinished, please click to continue reading for exciting follow-up content )

Serialized

Technical serialization: Application of Cre-loxP site-specific recombination system in the study of conditioned target gene function in mice

7.
Thoughts and suggestions in the application of Cre-loxP site-specific recombination system