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Review
. 2023 Jan 7;6(1):e1242.
doi: 10.1002/jsp2.1242. eCollection 2023 Mar.

Constitutive and conditional gene knockout mice for the study of intervertebral disc degeneration: Current status, decision considerations, and future possibilities

Ze-Yu Lu  1 , Peng-Bo Chen  1 , Qing-Yin Xu  1 , Bo Li  1 , Sheng-Dan Jiang  1 , Lei-Sheng Jiang  1 , Xin-Feng Zheng  1
Affiliations
Review

Constitutive and conditional gene knockout mice for the study of intervertebral disc degeneration: Current status, decision considerations, and future possibilities

Ze-Yu Lu et al. JOR Spine. .

Abstract

There have been an increasing number of patients with degenerative disc diseases due to the aging population. In light of this, studies on the pathogenesis of intervertebral disc degeneration have become a hot topic, and gene knockout mice have become a valuable tool in this field of research. With the development of science and technology, constitutive gene knockout mice can be constructed using homologous recombination, zinc finger nuclease, transcription activator-like effector nuclease technology and clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, and conditional gene knockout mice can be constructed using the Cre/LoxP system. The gene-edited mice using these techniques have been widely used in the studies on disc degeneration. This paper reviews the development process and principles of these technologies, functions of the edited genes in disc degeneration, advantages, and disadvantages of different methods and possible targets of the specific Cre recombinase in intervertebral discs. Recommendations for the choice of suitable gene-edited model mice are presented. At the same time, possible technological improvements in the future are also discussed.

Keywords: construction; gene edition technology; gene targeting; guideline; intervertebral disc degeneration; knockout; mouse model; specific Cre recombinase.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Steps of making knockout mice by homologous recombination (HR) principle. (A) Construction of gene vector. The target gene is recombined into a vector with marker genes such as neo or tk, so that it loses its physiological function and becomes a recombinant vector. (B) The vector is introduced into homologous embryonic stem (ES) cells in a certain way, and the exogenous DNA is recombined with the corresponding part of ES cell genome. (C) The hit cells are selected by positive and negative selection (PNS) or polymerase chain reaction (PCR). (D) Reconstruction is completed by blastocyst injection. (E) Chimeric animals are obtained after embryo transplantation, and then mating chimeric mice to obtain homozygous gene knockout mice. GFP, green fluorescent protein
FIGURE 2
FIGURE 2
Principles of gene knockout by zinc finger nuclease (ZFN), transcription activator‐like effector nuclease (TALEN), and CRISPR/Cas9 technologies. (A) Zinc finger (ZF) structures are assembled as modular assemblies. When ZF DNA‐binding domain can be perfectly paired with the target DNA sequence and FokI is dimerized, targeted site‐specific DNA is cut off. (B) The most common repeat‐variable di‐residues (RVDs), which can recognize four types of bases such as A, G, C, and T, respectively, are NI, NN, HD, and NG. The DNA specific recognition domain on TALE combines with the DNA sequence on the target site, and then FokI cutting domain is dimerized to cut off the specific sites of the target DNA sequence. (C) DNA double‐strand break (DSB) is implemented by ZFN, TALEN, and CRISPR/Cas9. (D) Cas9, crRNA, and tracrRNA jointly establish the Cas9 protein‐RNA mechanism. Cas9 targets virus or plasmid DNA by recognizing protospacer adjacent motif (PAM), and then cuts the DNA at the target site.
FIGURE 3
FIGURE 3
The location of Cre recombinase activation in the intervertebral disc (IVD). Some Cre mice target specific sites of the IVDs. Cre can be expressed in specific cells or tissue and the target gene at that site is knocked out. Therefore, the location of Cre recombinase activation in the IVD is critical. Cre recombinase activated in nucleus pulposus (NP): Col2 Cre , Col1 Cre , Shh Cre , Shh CreER , Acan CreER , Krt19 CreER , Noto Cre , Ella Cre , and Lepr Cre . Cre recombinase activated in inner annulus fibrosus (IAF): Col2 CreER and Scx Cre . Cre recombinase activated in outer annulus fibrosus (OAF): Col1 CreER , Lepr Cre , and Scx Cre . Cre recombinase activated in cartilaginous endplate (CEP): Col2 Cre , Col2 CreER , Col1 Cre , Shh Cre , Ella Cre , Acan CreER , and Osx Cre . Cre recombinase activated in growth plate (GP): Col2 Cre , Col2 CreER , Shh Cre , Osx Cre , and Acan CreER . Among them, the expression sites of Col1 Cre and Ella Cre , marked in rose red, have not been confirmed, but there have been phenotypic changes in IVDs in related experiments. Acan, aggrecan; Col1, type I collagen; Col2, type Il collagen; ER, estrogen receptor; Krt19, cytokeratin 19; Lepr, leptin receptor; Noto, notochord; Osx, osterix; Scx, Scleraxis; Shh, sonic hedgehog
FIGURE 4
FIGURE 4
Basic principle of conditional gene knockout using Cre/LoxP system. (A) When Flox mice inserted by two LoxP loci in the same direction at both ends of one or more important exons of the target gene are crossed with mice that express a tissue‐specific Cre recombinase, the progeny which contains both the Cre gene and the LoxP gene can knock out the target gene in a specific tissue or cells. (B) tamoxifen (TAM) is most commonly used as an inducer that binds to the fused mutant estrogen receptor (ER) on the Cre recombinase, replacing heat shock protein 90 (Hsp90), and induces the activation of the Cre/(ER)T system to knockout target gene. Appropriate timing of tamoxifen injection is selected according to the conditions of mice to avoid embryo or premature death.
FIGURE 5
FIGURE 5
A clear guideline to decision considerations for the use of knockout mice. First, you can select genes of interest by literature review, bioinformatics technology, or gene sequencing. The genes of interest were searched by NCBI gene ID and fully understood. Then you can search mouse models by going to The Jackson Laboratory's website. If it is a nonlethal gene, you can use homozygous KO for experiment. Instead, heterozygous KO mice or cKO mice could be selected for study. Sometimes cKO mice are not readily available, and you need to buy specific Cre mice and Flox mice to generate gene knockout progeny. However, sometimes site‐specific Cre mice cannot meet the needs of the disease background. At this point, you need to consider using CreER mice and Flox mice to breed offspring, and tamoxifen induction at specific stages to study disc degeneration.
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