Cre-loxp system and Viral vector (AAV and adenovirus)-based Cre tools (AAV-Cre and Ad-Cre)

Content index

Abstract

1. Introduction of Cre-loxP system

2. Application of inducible tissue-specific KO via Cre-loxP system

1) Inducible-tissue specific Cre-loxP system for in vivo and in vitro study

2) Tissue-specific AAV-Cre for in vivo study

3) Tissue/cell-specific Ad-Cre for in vivo or in vitro study

3. Cre-loxP induced overexpression: Cre-DIO system

Principle of Cre-DIO

Tissue-specific Cre mouse + AAV-DIO-GOI for tissue-specific gene overexpression

AAV-DIO in neuroscience

4. Summary

5. Reference

Product list: Cre/loxP tools in AAV vector(AAV-Cre), adenoviral vector(Ad-Cre) and lentiviral vector(Lv-Cre)

Cat No.	Pre-made Cre	Viral vector	Protomer	Promoter Tissue Specificity	Promoter Tissue Specificity characteristics
GMV-AdCre-01	Ad-CMV-Cre	Adenovirus	CMV	Total	Total
GMV-AdCre-02	Ad-CMV-Cre-GFP	Adenovirus	CMV	Total	Total
GMV-AAV9Cre-01	AAV9-CMV-Cre	AAV GeneMedi offer multiple serotypes of AAV-Cre loxP system for your option: · AAV1-Cre · AAV2-Cre · AAV2 variant (Y444F)-Cre · AAV2 variant (Y272F, Y444F, Y500F, Y730F)-Cre · AAV2 variant (Y444F, Y730F, Y500F, Y272F, Y704F, Y252F)-Cre · AAV2 variant(AAV2.7m8)-Cre · AAV5-Cre · AAV6-Cre · AAV8-Cre · AAV8-1m-Cre · AAV8-2m-Cre · AAV8 variant (Y733F, Y447F, Y275)-Cre · AAV9-Cre · AAV-Rh.10-Cre · AAV-DJ-Cre · AAV-DJ/8-Cre · AAV-Retro (Retrograde)-Cre · AAV-PHP.B-Cre · AAV9-PHP.A-Cre · AAV-PHP.eB-Cre · AAV-PHP.S-Cre	CMV	Total	Total
GMV-AAV9Cre-02	AAV9-CMV-Cre-ZsGreen		CMV	Total	Total
GMV-AAV9Cre-03	AAV9-CAG-Cre		CAG	Total	Total
GMV-AAV9Cre-04	AAV9-CAG-Cre-ZsGreen		CAG	Total	Total
GMV-AAV9Cre-05	AAV9-Syn-Cre-EGFP		synapsin (SYN)	Neuro	Neuron-specific promoter
GMV-AAV9Cre-06	AAV9-Syn-Cre		synapsin (SYN)	Neuro	Neuron-specific promoter
GMV-AAV9Cre-07	AAV9-CaMKII-Cre-EGFP		CaMKII	Neuro	Forebrain glutamate neuron-specific promoter
GMV-AAV9Cre-08	AAV9-CaMKII-Cre		CaMKII	Neuro	Forebrain glutamate neuron-specific promoter
GMV-AAV9Cre-09	AAV9-GFAP-Cre-EGFP		GFAP	Neuro	Astrocyte specific promoter
GMV-AAV9Cre-10	AAV9-GFAP-Cre		GFAP	Neuro	Astrocyte specific promoter
GMV-AAV9Cre-11	AAV9-cTNT-Cre		cTnT	heart	Cardiomyocyte specific promoter
GMV-AAV9Cre-12	AAV9-cTNT-Cre-EGFP		cTnT	heart	Cardiomyocyte specific promoter
GMV-AAV9Cre-13	AAV9-TBG-Cre		TBG	liver	Hepatic specific promoter
GMV-AAV9Cre-14	AAV9-TBG-Cre-ZsGreen		TBG	liver	Hepatic specific promoter
GMV-AAV9Cre-15	AAV9-TIE-Cre		TIE	endothelial	Endothelial-specific promoter
GMV-AAV9Cre-16	AAV9-cd68-Cre		cd68	Neuro	Microglia-specific promoter
GMV-AAV9Cre-17	AAV9-c-fos-Cre		c-fos	Neuro	Excitatory neuron promoter
GMV-AAV9Cre-18	AAV9-mecp2-Cre		mecp2	Neuro	Shorter neuron-specific promoter
GMV-AAV9Cre-19	AAV9-MHCK7-Cre		MHCK7	muscle	Muscle specific promoter
GMV-AAV9Cre-20	AAV9-Rpe65-Cre		Rpe65	retina	Retinal specific promoter
GMV-AAV9Cre-21	AAV9-sm22a-Cre		sm22a	muscle	Smooth muscle specific promoter
GMV-AAV9Cre-22	AAV9-Spc5-12-Cre		Spc5-12	muscle	Myocyte specific promoter
GMV-AAV9Cre-23	AAV9-Ta1-Cre		Tal	Neuro	Early neuron-specific promoter
GMV-LvCre-01	Lv-CMV-Cre	Lentivirus	CMV	Total	Total
GMV-LvCre-02	Lv-CMV-Cre-zsgreen	Lentivirus	CMV	Total	Total

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Abstract

Cre-loxP system is widely used in the field of biosciences, especially in the generation of genetically engineered mice (knockout or overexpression), enabling researchers to study the function of gene of interest (GOI). To date, numerous systems, derived from the basic Cre-loxP system, have been developed to precisely control gene expression at desired time or cells. Here, we briefly introduce Cre-loxP system, and give a summary about its applications, especially viral vectors-mediated Cre expression (such as AAV-Cre or Ad-Cre) for in vitro or in vivo studies.

1. Introduction of Cre-loxP system

As a powerful gene editing technology, Cre (cyclization recombinase) was first discovered in bacteriophage P1 to recognize and bind to the specific DNA sequences, named as loxP (locus of crossing over, x, P1) site and thus mediate site-specific rearrangement of target DNA sequences between two loxP sites (Fig. 1A), which can be located in the same or separate DNA fragments [1]. In this recombination process via Cre-loxP system, there were three rearrangement outcomes based on the orientation and location of the two loxP sites, which were shown in Fig. 1: ① gene deletion would happen, if the two loxP sites locate in the same DNA strand as well as the same orientation (Fig. 1B); ② gene inversion would happen, if the two loxP sites locate in the same DNA strand but in opposite orientations (Fig. 1C); ③ sequence translocation would happen, if the sites are in separate DNA molecules (Fig. 1D) [2, 3].

Figure 1. Site-specific recombination via Cre-loxP system. (A) Close-up of Cre recombinase-mediated recombination between two 34 bp loxP sites, which consists of two 13 bp inverted and palindromic repeats (sequences in red or purple) and 8 bp core sequences (sequences in blue, “N” indicates the bases may vary from the canonical sequence of GCATACAT), which determines the orientation of the loxP site [4]. (B-D) Schematic of Cre recombinase mediated target gene deletion (B), inversion (C), or translocation (D).

To date, Cre-loxP system has been widely applied in the generation of gene knockout (KO) mice [5] (Fig. 2), especially the inducible tissue-specific gene KO mice [6]. First, loxP sites should be introduced into the introns to flank an essential exon of targeted DNA (floxed DNA) in murine embryonic stem cells (mESCs) through homologous recombination, which will not affect the expression level of target gene. Then, mESCs need to be injected into a pseudopregnant female mouse blastocyst to develop into a whole mouse carrying the desired targeted mutation. After breeding with the mouse containing Cre gene, the heterozygous mice with target gene excision will be obtained (Fig. 2).

Figure 2. Generation schematics of progeny mice with gene of interest (GOI) mutation via Cre-loxP technology. In principle, once one mouse carrying floxed DNA breeds with another mouse carrying Cre gene, their offspring will have both flanked DNA and Cre. Thus, Cre recombinase will excises floxed loci and inactivates GOI [7]. GOI, gene of interest.

2. Application of inducible tissue-specific KO via Cre-loxP system

To figure out the gene regulation and function more precisely, several sophisticated inducible systems were introduced to control the expression of Cre at specific time (temporal) and tissue (spatial) in vivo or in vitro [7]. There are several common inducible systems via cell-specific regulatory elements (such as promoters or enhancers), exogenous inducers (such as tamoxifen (tam) or tetracycline (tet)) [4, 7-10], and viral vectors (such as adeno-associated viral (AAV) vectors) [11].

1) Inducible-tissue specific Cre-loxP system for in vivo and in vitro study

a) Tissue-specific promoter driven Cre-loxP system for conditional knock-out (KO)

To achieve the conditional knock-out (KO) of gene of interest (GOI) in specific tissue or organ, cell-specific promoter/enhancer driven Cre lines are used to breed with GOI-loxP lines. Their offspring will get both cell-specific driven Cre and floxed DNA (GOI), resulting in tissue-specific GOI excision (Fig. 3). A great number of cell-specific promoters or enhancers have been developed, such as Lyz2 driven Cre specific in macrophage, and CD45 driven Cre specific in hematopoietic tissues. Some examples are listed in the following Table 1.

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Figure 3. Generation schematics for conditional knock-out (KO) using tissue-specific Cre/loxP tool. In principle, one mouse with tissue (X)-specific driven Cre gene breeds with another mouse with floxed GOI, producing offspring carrying floxed GOI Y and Cre recombinase in tissue X. Thus, GOI Y would be excised by Cre in tissue X [7]. GOI, gene of interest.

Table 1. Examples of tissue-specific promoter driven Cre-loxP system
Tissue/organ	cell	Promoter/enhancer	Reference
Cerebrum	Neuronal and glia cell precursors	Aldh1l1	[12]
	Cerebrum	CaMIIα	[13]
	GABAergic interneurons	Dlx1	[14]
	GABAergic neurons	Dlx5/6	[15-17]
	DRG	Gad2	[18]
	Astrocyte	GFAP	[19, 20]
	CA3 pyramidal cells	Grik4	[21]
	Arcuate nucleus	Lepr	[22]
	Neuronal and glia cell precursors	Nes	[23]
	DRG neurons	nNOS	[18]
	Retinal Müller glial cells	Pdgfrα	[24]
	Myelinating cells	PLP1	[25]
	DRG neurons	Pv (Pvalb)	[18]
	GABAergic neurons	Slc17a6	[26]
	GABAergic neurons	Sst, Vip	[17]
Cerebellum	Cerebellar Purkinje cells	Pcp2	[27]
Brain stem	Dopamine and serotonin neurons	Slc6a3 (DAT)	[28]
	Serotonin neurons	ePet (Fev)	[29]
	Vagal sensory neurons	Npy2r	[30]
Spinal cord	Mechanosensory dorsal horn	Cdh3, Htr6	[31]
Skin	Basal layer of the epidermis	Krt5	[32]
	Epidermis keratinocytes	Krt10	[33]
	Epidermal progenitors	Krt18	[34]
Immune system	Macrophage	Lyz2	[35]
	Dendritic cells	CD11c (Itgax)	[36]
	Mast cells	CMA1	[37]
	T cells	CD2	[38]
		CD4	[39]
		CD8a	[40]
		Foxp3	[41]
		Lck	[42]
		OX40	[43]
	B cells	CD19	[44, 45]
	Hematopoietic cells	CD15	[46]
Musculoskeletal	Osteochondro progenitors	Twist2 (Dermol)	[47, 48]
	Osteochondro progenitors	Prrx1	[49]
	Osteoblasts	BGLOP	[50]
	Osteocytes	Dmp1	[51]
	Osteoclasts	Ctsk	[52]
	Cardiomyocytes	NKX2.5	[53]
	Muscle	ACTA1 (HSA)	[54]
Digestive system	Stomach	ATP4b	[55]
	Intestine	Car1	[56]
	Intestine	Vil1	[57]
	Liver	Alb	[58, 59]
	PanCreas	Ghrl	[60]
		Ins1 (MIP)	[61]
		Ins2 (RIP)	[62]
		Ptf1a	[63]
Urogenital system	Bladder	Upk2	[64]
	Kidney	Aqp2	[65]
		Gdnf	[66]
		Ggt1	[67]
	Ovary	Gdf9	[68]
	Ovary	Zp3	[69]
	Testis	Amh	[70]
	Testis	Hspa2	[71]

b) Inducible Cre-loxP system

One inducible system is tamoxifen-inducible Cre-loxP system (Fig. 4A). When ubiquitous or tissue-specific promoter driven Cre is fused with the estrogen receptor containing a mutated ligand binding domain (ER-LBD), which is normally bound by heat shock protein 90 (HSP90), Cre-ER-HSP90 complex would be prevented from entering the nucleus. Once bound by the hormone (such as estrogen) or synthetic analogs (such as tamoxifen, T, or 4-hydoxytamoxifen, 4-OHT), ER will be released from HSP90. This enables the nuclear translocation of Cre-ERT complex and the following interaction between Cre and loxP sites [8, 32].

Another inducible system is tetracycline inducible Cre-loxP system, also known as doxycycline (dox; a tetracycline derivative)-inducible Cre system. This system has two mode: tet-on (Fig. 4B) and tet-off (Fig. 4C), responsible for the activation and inactivation of Cre gene, respectively [72, 73]. There are three elements orchestrating to control Cre expression in this system: reverse tetracycline-controlled transactivator (rtTA), tetracycline-controlled transactivator (tTA) and tetracycline responsive element (TRE), also named as a tetracycline operon (TetO). In tet-on system, ubiquitous or tissue-specific promoter driven rtTA binds to dox to turn on the transcription of Cre gene (Fig. 4B). On the contrary, in tet-off system, tTA binds to dox to turn off the transcription of Cre gene (Fig. 4C).

Figure 4. Operational principles of inducible tissue-specific Cre-loxP system [7]. (A) Tamoxifen-inducible Cre-loxP system with estrogen receptor (ER) fused to Cre (CreER). When tamoxifen is supplemented to bind to ER, Cre will translocate into nucleus to mediate the excision target gene Y. (B) Tetracycline (Tet)-on Cre-loxP system. Without dox, inactivated rtTA cannot activate the tetO7 (7 repeats of a 19 nuclotide tetO minimal promoter) for Cre expression. Upon the supplementation of dox, activated rtTA binds to tetO7 to initiate the transcription of Cre gene. (C) Tet-off Cre-loxP system. In the absence of dox, tTA is able to bind to tetO7 to activate Cre expression. Following the administration of dox, tTA will be silenced as well as lose the ability to bind to tetO7 and activate Cre transcription.

So far, a huge number of Cre transgenic lines have been established to combine with GOI loxP lines to generate conditional knockout mice or cell lines at the desired period and tissues in vitro or in vivo. Given that it is time and labor consuming to use Cre transgenic mice lines, viral vectors are convenient and versatile tools for the expression of Cre in certain cells or tissues, such as adeno-associated viral (AAV) vectors, as well as cell lines, such as adenovirus (Ad).

2) Tissue-specific AAV-Cre for in vivo study

As the most excellent gene therapy vector, recombinant AAV can mediate long-term stable expression of target genes in vitro/vivo with broad range of host and low immunity. Over the past decades, numerous AAV serotypes have been discovered or developed, and each serotype has its special tropism. For instance, AAV2 can moderately transduce several tissue types, including central nervous system, liver, muscle, and lung. Similarly within the CNS, AAV1 and AAV5 show higher transduction frequencies than AAV2 in all injected regions [74, 75], while AAV4 just appears to transduce specific cell types, such as the astrocytes and ependyma in the subventricular zone [76]. In skeletal muscle cells, AAV1, AAV6 and AAV7 is reported to present very high transduction rate [77-80], while AAV8 exhibits transduction ability with super efficiency not only in hepatocytes but also in other organs [78, 81, 82]. In addition to skeletal muscle cells, AAV6 has been also reported to mediate more efficient transduction of airway epithelial cells in mouse lungs compared to that of AAV2 [81], which may supply AAV6 with more significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis considering the lower immunogenicity of AAV6 than of AAV2 [78, 82].

GeneMedi holds the expertise at AAV production, you can find more information and protocols about AAV on this website: https://www.genemedi.net/i/aav-packaging.

Combined with Cre-loxP system, AAV containing Cre (AAV-Cre) can help mediate GOI conditional KO in specific tissues or organs based on its special tissue trophism (Fig. 5). This technology has been widely applied into several research fields, especially for the gene function studies in neurosciences. Some of examples are listed in Table 2.

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Figure 5. Generation schematics for AAV-mediated tissue-specific GOI deletion. About two weeks post-infection of AAV-Cre viruses, the transgenic mice with floxed DNA will express Cre in specific tissue based on the AAV tropism. Then, in this specific tissue, Cre recombinase will bind and excise the loxP sites, thus inactivate the GOI.

Table 2. Examples of tissue-specific KO via AAV-Cre/loxP system
AAV serotype	Tissue/organ	Delivery route	Gene	Reference
AAV9	Hippocampus or central amygdala	Stereotaxic microinjection	IRs and IGF1Rs	[11]
AAV9	Astrocyte	In vitro transduction	IGFR	[83]
AAV2/5	Large DRG neurons	Intrathecal injection	Na(V)1.6	[84]
AAV9	Spinal dorsal horn neurons	Intrathecal injection	SIRT1	[85]
AAV2	Cortical neurons	Unilateral intracortical injections	PTEN	[86]
AAV5	Forebrain excitatory neurons	Stereotaxic microinjection	SIRT1	[87]
AAV2/1	Muscle	Intramuscular injection	Mtm1	[88]
AAV2	Ventral tegmental area, VTA	Intra-VTA infusion	Gabrd	[89]
AAV8	Sensorimotor cortex, hippocampus, and spinal cord	Intra-sensorimotor cortex injection	PTEN	[90]
AAV2/5	mesolimbic Ventral tegmental area (VTA), NAc core and central amygdala (CeA)	Intracerebroventricular (ICV) injection	Lepr	[91]
AAV9	Dentate gyrus (DG)	Intra-DG injection	Shh	[92]
AAV8	Liver	Lateral tail vein injection	HKDC1	[93]
AAV1	Hypothalamus	Stereotaxic microinjection	AC3	[94]

Additionally, tissue-specific promoter is also used to drive Cre expression to strengthen the tissue specificity of GOI knockout. Several tissue-specific promoters, within the loading capacity of AAV vectors, are listed in the following table 3.

Table 3. Tissue-specific promoters in AAV-Cre
Tissue/organ	Promoter	Specificity	Promoter size	Species
Neurocyte	hSyn	Neuron	0.5kb	human
	mecp2	Short neuron	0.2kb	mouse
	TUBA1A	Developing neuron	1.2kb	human
	c-fos	Excitatory neuron	1.7kb	mouse
	CaMKIIa	Glutamate neurons in the forebrain	1.1kb	mouse
	hVGAT	GABAergic interneurons	1.8kb	human
	Slc6a3	Dopamine and serotonin neurons	1.2kb	mouse
	gfaABC1D	Astrocyte	0.9kb	human
	Iba1	Keratinocyte	1.5kb	mouse
	CNP	Schwann cell	1.5kb	mouse
Muscle	cTNT	Cardiomyocyte	0.7kb	chicken
	mNKX2.5	Cardiomyocyte	1.2kb	mouse
	SM22a	Smooth muscle	0.5kb	mouse
	MCK	Muscle	1.3kb	mouse
	MYOG	Myoblast	1.4kb	mouse
	ACTA1	Myocyte	1.2kb	mouse
Skeleton	COL2A1	Chondrocyte	1.2kb	mouse
Skeleton	mRUNX2	Osteoblast	1kb	mouse
Lung	SP-C	Lung epithelial cells	0.3kb	mouse
Liver	TBG	Liver cells	0.4kb	human
PanCreas	PDX1	PanCreas cells	1.2kb	mouse
Fat	FABP4	Fat cells	1.6kb	mouse
Endothelial	TIE	Endothelial cells	1.2kb	mouse
Corneum	K14	Keratinocyte	1.5kb	mouse
Retina	rpe65	Retinal cells	0.7kb	mouse

GeneMedi offers kinds of pre-made AAV-Cre particles in different serotypes driven by optional promoters. Click here for more information about GeneMedi’s ready-to-use AAV-Cre:https://www.genemedi.net/i/Cre-products

3) Tissue/cell-specific Ad-Cre for in vivo or in vitro study

Based on human adenovirus type 5 (Ad5), recombinant adenovirus (Ad) a replication-defective adenoviral vector system, is widely used for gene delivery in most cell types [95], and Genemedi got a rich experience in adenovirus packaging, you could find more information on https://www.genemedi.net/i/adenovirus-packaging.

Combined with Cre-loxP system and cell-specific promoter driven Cre, Adenoviral vector can also help mediate GOI conditional KO in vivo. Besides, Adenoviral vector containing Cre (Ad-Cre) also exhibits great advantages and high efficiency for the in vitro studies, especially for the transduction of primary cells [96], which are commonly difficult to be transduced (Fig. 5). As a promising and efficient tool, Ad-Cre system has been extensively used into the research field of cell lineage tracing and fate mapping. Some of examples are listed in Table 4.

Table 4. Examples of Ad-Cre-loxP system
Promoter	Tissue/organ/cell	Delivery route	Gene	Reference
Keratin 8/ Keratin 5	Mammary epithelial cells	Intraductal injection	YFP	[97]
Keratin 8/ Keratin 14	Mammary epithelial cells	Intraductal injection	YFP	[98]
——	Leydig cells	Intratesticular injection	Gata4 and Gata6	[99]
CMV	Subfornical organ-hypothalamic-hypophysial and brain stem-parabrachial axes	Stereotaxic microinjections	β-galactosidase (β-gal)	[96]
CMV	Vasopressin-synthesizing magnocellular neurons	Retrograde transport	β-galactosidase (β-gal)	[96]
CMV	Primary neuronal cells	In vitro inoculation	β-galactosidase (β-gal)	[96]
——	PASMCs	In vitro inoculation	ALK2	[100]
——	Muscle	Retropopliteal injection	ALK2	[100]
CAG	HepG2 cell line	In vitro inoculation	SV40Tag	[101]
——	Primary osteoblasts	In vitro inoculation	IGF-IR	[102]
——	Mesangial cells	In vitro inoculation	EP4	[103]
——	Primary calvarial cells	In vitro inoculation	IGF-IR	[104]

GeneMedi offers kinds of pre-made Adenovirus-Cre (Ad5-Cre, Ad-Cre) particle driven by optional promoters. Click here for more information about GeneMedi’s ready-to-use Adenovirus-Cre (Ad5-Cre, Ad-Cre):https://www.genemedi.net/i/Cre-products

3. Cre-loxP induced overexpression: Cre-DIO system

Besides GOI deletion, Cre-loxP system also shows great advantages in GOI overexpression, such as Cre-dependent GOI expression, and Cre-DIO (double-floxed inverse open reading frame) system. 1) Cre-dependent GOI expression: a stop codon between two loxP sites (loxP-stop-loxP, LSL, floxed stop) was placed at the upstream of GOI, thus preventing the expression GOI. Once Cre is supplemented, the stop codon and one lox site would be excised, and the GOI expression would be activated [105]. 2) Considering that the three modes of Cre-loxP technology are all reversible, Cre-DIO system were created to induce inverse gene expression utilizing two pairs of lox sites [106, 107].

Principle of Cre-DIO

Although Cre-loxP tool is widespread in biological sciences, it is really restrictive to achieve more than one recombination event in a cell. Therefore, numerous loxP mutant versions have been created, with the variation from the unique 8 bp core asymmetric spacer "NNNTANNN", such as loxN (GtATACcT), lox2272 (GgATACtT), lox5171 (ATGTGTaC), loxM2 (AgaaAcca), loxM3 (taaTACCA), loxM11 (AGATAGAA), and lox511 (GtATACAT). These lox variants only proceed recombination with the same type of lox sites, with no ability to interact with the other types. In Cre-DIO system, there are two pairs of lox sites (lox site 1 and lox site 2) flanking inverted GOI and reporter gene. In the absence of Cre, the inverted GOI and reporter are inactivated. Upon administration of Cre, the two pairs of lox sites will undergo recombination, resulting in the expression of GOI and reporter gene. Simultaneously, the cells with recombination interaction are labeled by the reporter gene (Fig. 6). The Cre-DIO tool utilizes initially inverted GOI to truly inactivate GOI, and activates GOI after a series of recombination interactions. Compared to the traditional overexpression strategy via floxed stop, this technology is much more effective to avoid the transcriptional leakage when the stop codon fails to totally suppress the transcription of GOI.

Figure 6. Principle of Cre-DIO (double-floxed inverse open reading frame) system. Without Cre, the GOI and reporter gene are inverted and silenced. In the presence of Cre, first, the transgene will be reoriented, then one of the two lox sites in each pair will be excised. Finally, the transgene contains only an incompatible pair of lox sites (lox 2-GOI-Reporter-lox 1) that are no longer responsive to Cre. Thus, the GOI and reporter gene will be activated. Lox site 1 and lox site 2 are variants of lox sites.

Tissue-specific Cre mouse + AAV-DIO-GOI for tissue-specific gene overexpression

To facilitate the use of Cre-DIO system, AAV vectors have been introduced to tissue-specific Cre mouse to mediate tissue-specific gene overexpression. Without injection of AAV vector carrying DOI-GOI system (AAV-DIO-GOI), tissue X-specific Cre mouse cannot express GOI and reporter gene in tissue X. After injection of AAV-DIO-GOI into tissue X, Cre recombinase will interact with lox sites, thus activating the expression of GOI and reporter. Synchronously, the tissue X will be labeled by the reporter gene (Fig. 7). This system utilizes high cell-specific promoter to drive Cre recombinase expression and a strong promoter to drive GOI expression, which ideally occur just at the appropriate time point and specific cells. As a powerful system with high spatiotemporal fidelity, AAV-DIO exhibits great advantages and potential in the study of neural circuits and networks, behavior, specific cell populations (lineage tracing), animal models of disease, as well as in high-throughput ex vivo researches.

Figure 7. Principle of tissue-specific gene overexpression via AAV-DIO-GOI with tissue-specific Cre mouse. Without AAV-DOI-GOI, the GOI and reporter gene do not express in tissue X. Upon AAV-DOI-GOI administration into the mouse with tissue X-specific Cre, the GOI and reporter gene will be activated in tissue X and the tissue X will be labeled by reporter gene (green). Lox site 1 and lox site 2 are variants of lox sites.

AAV-DIO in neuroscience

In the research field of neuroscience, AAV-DIO shows outstanding spatiotemporal fidelity and is perfectly suitable for examining and manipulating neuronal function in vivo, such as studies in neural circuits and networks, behavior and so on. Optogenetics integrates optics with genetic engineering to measure or manipulate the biomolecular processes, especially in neuroscience [108]. By delivering optical signals with specific wavelength to precisely detect, measure, or control biological processes in living tissues, optogenetics opens up new landscapes for the study of biology, both in health and disease [109]. With the combination of AAV-DIO and optogenetics, researchers control and monitor the activities of individual neurons, and precisely measure the manipulation effects in real-time. For example, in Fig. 8, with AAV-DIO system, the neuron X specifically express opsin (such ChR2) and is labeled by the reporter gene (such as mCherry), but remains silenced. When exposed to light in correct wavelength, neuron X will be activated (Fig. 8) [110].

Figure 8. Principle of optogenetics tool in combination with AAV-DIO. With no light in specific wavelength matched to opsin, such as ChR2, ChR2 gene and the reporter gene is specifically expressed in neurons (location in red). Once exposed to light with the matched wavelength, ChR2 cation channels will be open and Na+ flow into neural cells. Thus, the neuron will be activated.

Genemedi possesses a complete library of optogenetics components into serotypes of AAV vector, which is listed in table 5. You can find more information on this website: https://www.genemedi.net/i/aav-optogenetics.

Table 5. List of optogenetics tools in AAV
AAV Plasmids	Promoter and Expression Characteristics	Fluorescent Protein Label	Cre dependent	Optogenetics effect
pAAV-CAG-DIO-eNpHR3.0-EYFP	CAG general and strong expression	EYFP	yes	Inhibition
pAAV-CAG -DIO-hChR2(H134R)-mCherry		mCherry	yes	activation
pAAV-CAG-DIO-ArchT-EYFP		EYFP	yes	inhibition
pAAV-CAG -DIO-C1V1 (t/t)-TS-mCherry		mCherry	yes	activation
pAAV-CAG-DIO-Arch3.0-EYFP		EYFP	yes	inhibition
pAAV- CAG -DIO-hCHETA-EYFP		EYFP	yes	activation
pAAV-CMV-DIO-eNpHR3.0-EYFP	CMV(general and strong expression)	EYFP	yes	inhibition
pAAV-CMV-DIO-hChR2(H134R)-mCherry		mCherry	yes	activation
pAAV-CMV-DIO-ArchT-EYFP		EYFP	yes	inhibition
pAAV-CMV-DIO-C1V1 (t/t)-TS-mCherry		mCherry	yes	activation
pAAV-CMV-DIO-Arch3.0-EYFP		EYFP	yes	inhibition
pAAV-CMV-DIO-hCHETA-EYFP		EYFP	yes	activation
pAAV-hSyn-eNpHR3.0-EYFP	hSyn neuron specific expression	EYFP	No	inhibition
pAAV-hSyn-hChR2(H134R)-mCherry		mCherry	No	activation
pAAV-hSyn-ArchT-EYFP		EYFP	No	inhibition
pAAV-hSyn-C1V1-(t/t)-TS-mCherry		mCherry	No	activation
pAAV-hSyn-Arch3.0-EYFP		EYFP	No	inhibition
pAAV-hSyn-DIO-hCHETA-EYFP		EYFP	yes	activation
pAAV-GFAP-eNpHR3.0-EYFP	GFAP astrocyte specific expression	EYFP	No	inhibition
pAAV-GFAP-hChR2(H134R)-mCherry		mCherry	No	activation
pAAV-GFAP-ArchT-EYFP		EYFP	No	inhibition
pAAV-GFAP-C1V1 (t/t)-TS-mCherry		mCherry	No	activation
pAAV-GFAP-Arch3.0-EYFP		EYFP	No	inhibition
pAAV-GFAP-hCHETA-EYFP		EYFP	No	activation
pAAV-CaMKII-eNpHR3.0-EYFP	CaMKII neuron specific expression	EYFP	No	inhibition
pAAV-CaMKII-hChR2(H134R)-mCherry		mCherry	No	activation
pAAV-CaMKII-ArchT-EYFP		EYFP	No	inhibition
pAAV-CaMKII-C1V1 (t/t)-TS-mCherry		mCherry	No	activation
pAAV-CaMKII-Arch3.0-EYFP		EYFP	No	inhibition
pAAV-CaMKII-hCHETA-EYFP		EYFP	No	activation

4. Summary

The Cre-loxP system, especially inducible tissue-specific knockout and viral vector-mediated Cre expression, has been highly utilized in genetics and cell biology research. Nevertheless, it seems that Cre-loxP system will continue to be prevalent in current and future research studies. GeneMedi is proficient in viral vector development and offers kinds of viral vector-based Cre tools, we can provide the best services and products if required.

5. References

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