RU2849189C1 - Line of transgenic mice c57bl/6j-tg(cmv-h2b-egfp)12ched, expressing fluorescently labelled histone h2b in postnatal age, and method for obtaining it - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: C57BL/6J-Tg(CMV-H2B-eGFP) transgenic mouse line. This line expresses fluorescently labelled histone H2B in postnatal age and is transformed with a transgenic cassette consisting of a CMV promoter, an SV40 polyadenylation signal, terminating transcription from said promoter, and an open reading frame of the chimeric H2B-eGFP gene with the sequence SEQ ID NO: 1. The invention also relates to a method for producing said line.

EFFECT: to provide a line of transgenic mice expressing the H2B-eGFP gene in a tissue-specific and age-dependent manner.

4 cl, 2 dwg, 3 ex

Description

The group of inventions relates to the field of molecular biotechnology and concerns a line of transgenic mice expressing fluorescently labeled proteins for marking cellular components.

The study of biological processes in vivo is a fundamental field of modern biology and medicine. Various methods are used to visualize and localize cells, proteins, and other biomolecules in living organisms, including specific staining of specific organelles or cellular compartments based on biochemical differences, fluorescent or bioluminescent labeling of specific proteins, and immunostaining.

However, these methods have their drawbacks, including the inability or non-specificity of identifying these components during the imaging stage. For example, some cellular components may exhibit bioluminescence or autofluorescence. This phenomenon is typical for fat deposits in the brain, which primarily fluoresce in the green spectrum (1).

When staining cells and biomolecules, achieving exceptional labeling specificity is difficult. For example, the well-known nucleic acid dye DAPI labels DNA well, but there is extensive evidence of its interaction with RNA (2).

Fluorescent labels, which absorb light of a specific wavelength and emit light of a different, longer wavelength, are among the most advanced imaging tools. This property allows them to be visualized using fluorescence microscopy. One way to use fluorescent labels in living organisms is to express fluorescent proteins in genetically modified animals. This includes creating variants of the body's endogenous proteins fused with fluorescent domains, which allows for the localization of the protein of interest during imaging. Using various genetic constructs allows for the specific expression of such proteins in certain cell types and tissues.

H2B is a histone protein that is a key component of nucleosomes in eukaryotic cells. Labeling H2B with a fluorescent protein creates a powerful tool for visualizing chromatin dynamics and chromosome behavior in living cells using a fluorescence microscope. Visualizing H2B-bound chromosomes enables the study of processes such as cell replication, repair, and division, cell cycle dynamics, chromosome organization, and the effects of genetic or chemical factors on chromatin structure.

Currently, many animal and human cell lines have been described that express exogenous H2B labeled with various fluorescent proteins (3-5).

Transgenic mouse strains expressing exogenous H2B labeled with various fluorescent proteins, such as eGFP (enhanced green fluorescent protein), mCherry (red fluorescent protein), and YFP (yellow fluorescent protein), are known. These proteins are widely used for visualizing cellular processes in vivo due to their ability to absorb light at one wavelength and emit at another, allowing for high-resolution monitoring of cell and protein dynamics in real time. For example, the H2B-eGFP mouse strain allows for the study of chromatin dynamics in living cells, particularly during cell division and DNA repair, providing a detailed picture of the cell cycle. Mouse strains expressing H2B-mCherry or H2B-YFP have also found application in multispectral analysis and simultaneous visualization of multiple cellular components (6, 7).

Disadvantages of existing transgenic mouse models include their non-specific and constant expression of fluorescent protein in all tissues and throughout the animal's life. This results in high background signal, complicating the precise visualization of specific cell types or tissues, especially when studying adult mice. Some models also do not provide sufficient nuclear fluorescence for direct visualization, requiring the use of additional dyes or antibodies. This complicates experimental procedures and can negatively impact cell or tissue viability. Taken together, these limitations reduce the effectiveness and versatility of existing transgenic models for specific studies, particularly those involving endothelial cells in adult animals. Furthermore, existing transgenic models are not readily available in Russia. Therefore, the development of new transgenic mouse models with preferential expression of fluorescent protein in endothelial cells in adult animals is a pressing challenge for the development of modern pharmacology in Russia.

The closest analogue to the claimed group of inventions—the prototype—is a transgenic mouse line (model) expressing the H2B-eGFP fusion protein under the control of the CAG (cytomegalovirus enhancer/chicken β-actin promoter) promoter. H2B-eGFP expression in this mouse line occurs constitutively in many tissues and at various stages of development, including the embryonic and postnatal periods. This model is widely used to visualize chromatin dynamics, cell division processes, and to study chromosome organization in living cells in vivo. This mouse line allows researchers to observe the behavior of nuclear material during development, as well as under various physiological and pathological conditions.

The method for obtaining this model involves microinjecting the resulting genetic construct into fertilized mouse eggs, transferring the microinjected eggs into the oviducts of pseudo-pregnant recipient females, selecting mice carrying the target mutation, crossing the latter with wild-type mice to obtain heterozygous females or mutant males carrying the target mutation, and crossing heterozygous mice from the previous stage to obtain the final line of mice expressing the H2B-eGFP fusion protein (5).

The disadvantages of the prototype model include its non-specific and constant expression of the fluorescent protein in all tissues and throughout the animal's life. This results in a high background signal, complicating the precise visualization of specific cell types or tissues, especially when studying adult animals. Furthermore, the lack of preferential expression in endothelial cells in adult animals limits the application of this model in studies of the vascular system, angiogenesis, and endothelial function.

The objective of this invention is to create a line of transgenic mice expressing the H2B-eGFP gene in a tissue-specific and age-dependent manner.

This task is accomplished using the C57BL/6J mouse strain. Zygotes obtained by in vitro fertilization are injected with a solution of a linear double-stranded DNA fragment encoding the chimeric H2B-eGFP gene under the control of the CMV promoter (CMV-H2B-eGFP). This transgene cassette contains the human histone 2B (H2B) gene sequence, the stop codon of which is replaced with a nucleic acid sequence encoding the eGFP protein. Expression of this fusion gene is controlled by the CMV promoter. This transgene cassette, due to its involvement in cellular repair processes, can be introduced into a random location in the zygote's genome. The embryos are then cultured to the two-cell stage and transferred to a recipient mouse, which carries and gives birth to potentially transgenic offspring.

Technical result: Obtaining a line of transgenic mice that carry in a homozygous state the integration of the CMV-H2B-eGFP transgenic cassette and express the H2B-eGFP gene contained therein in a tissue-specific and age-dependent manner, namely, its ubiquitous expression in perinatal age, and a decrease in expression by adulthood with the preservation of expression in highly specific cell types, primarily in endothelial cells (lining of blood vessels).

The essence of the invention

The invention involves a mouse strain expressing the H2B-eGFP gene in a tissue-specific and age-dependent manner. Specifically, during the perinatal period, expression of the transgene cassette is detected almost ubiquitously across a broad spectrum of cell types, while in adulthood, expression is observed in a narrow spectrum of specific cell types, primarily in the endothelium. The specific pattern of H2B-eGFP gene expression enables visualization of blood vessel capillaries in adult transgenic animals in vivo due to the fluorescent signal of endothelial cell nuclei. This opens up opportunities for studying endothelial cell biology, vascular growth dynamics, and wound healing, as well as for tracking changes influenced by various factors.

Furthermore, expression of the fusion protein is controlled by the CMV promoter (unlike the CAG promoter in similar animals (7)), allowing the transgenic animal to be easily identified at an early age by the presence of a fluorescent signal. At the age of up to one month, the fluorescent signal is detected in the nuclei of a wide range of organs and tissues, suggesting that H2B-eGFP expression is constitutive in animals during the perinatal period.

Then, probably due to the position effect, expression of the transgene construct in animals at the age of entering sexual maturity disappears, and at a later age the fluorescent signal is detected only in the nuclei of a highly specific number of cell types, mainly endothelial cells.

The combination of these factors makes the proposed transgenic mouse line unique and significant. This mouse line can be effectively used to study endothelial cell biology, develop new treatments for diseases associated with endothelial dysfunction, and test pharmaceuticals. These aspects make the model an important tool in research aimed at understanding disease mechanisms and developing new therapeutic approaches.

The method for generating the C57BL/6J-Tg(CMV-H2B-eGFP)12Ched transgenic mouse line consists of two stages. In the first stage, the genetic construct used subsequently for transgenesis is obtained. The H2B-GFP plasmid, containing the protein-coding portion of H2B fused to eGFP under the CMV promoter (Addgene No. 11680), was used as the initial construct. The plasmid was linearized using bacterial restriction enzymes that hydrolyze specific DNA sites flanking the transgene cassette sequence in the H2B-GFP plasmid. The enzymatic hydrolysis reaction products were then separated by length using agarose gel electrophoresis, and the 2006 bp product was isolated. was isolated and purified using a DNA extraction kit from agarose gel on sorbent columns, after which the DNA concentration in the resulting solution was determined using Nanodrop 2000. As a result, the CMV-H2B-eGFP transgene cassette was obtained, which is a double-stranded DNA fragment containing the chimeric H2B-eGFP gene under the control of the CMV promoter (CMV-H2B-eGFP).

The CMV-H2B-eGFP transgene cassette, containing the chimeric H2B-eGFP gene having the nucleotide sequence SEQ ID NO: 1, is characterized by the following features:

- is a double-stranded DNA molecule;

- has a size of 2006 bp;

- encodes the chimeric H2B-eGFP gene under the control of the transcription enhancer and promoter of CMV and the transcription terminator of the SV40 virus;

The graphical map of the cassette is shown in Fig. 1, and the nucleotide sequence SEQ ID NO: 1 is presented in the sequence listing.

In the second stage, the obtained CMV-H2B-eGFP transgene cassette was microinjected into the pronucleus of a fertilized egg, using the technology (8), into C57BL/6J mice, and potentially transgenic zygotes were obtained. Then, the surviving zygotes (two-cell embryos) were transplanted into pseudo-pregnant recipient females with a copulatory plug after mating with vasectomized males, using the technology (9). Before transferring the embryos into the pseudo-pregnant female, the latter were cultured for at least 12 hours in a CO ₂ incubator under mineral oil in KSOM-AA nutrient medium. Next, after the transfer of two-cell embryos, newborn mice were obtained, from which mice carrying the CMV-H2B-eGFP transgene cassette were selected using phenotyping by fluorescence of cell nuclei. To phenotype young animals, ear fragments were collected during standard animal tagging procedures upon separation from their mothers at three weeks of age. The ear fragments were analyzed using the ZOE imaging system in the eGFP fluorescence detection channel.

To obtain heterozygotes carrying the CMV-H2B-eGFP transgene cassette, the resulting mice were crossed with wild-type mice. The resulting heterozygous mice were then crossed with each other to produce homozygous mice and the final line of mice transgenic for the CMV-H2B-eGFP cassette. Genotyping for the presence of the CMV-H2B-eGFP construct was performed by detecting the GFP fluorescent signal from cell nuclei using a fluorescence microscope (Figure 2).

As a result, a new line of transgenic mice C57BL/6J-Tg(CMV-H2B-eGFP)12Ched was created, characterized by tissue-specific and age-dependent expression of the H2B-eGFP gene, namely its ubiquitous expression in perinatal age, and a decrease in expression by adulthood with the preservation of expression in highly specific cell types, mainly in endothelial cells (lining of blood vessels).

The invention is illustrated by the following figures.

Fig. 1. Graphic map of the CMV-H2B-eGFP cassette providing high expression of the H2B-eGFP reporter protein in transgenic animals.

Fig. 2. Visualization of the fluorescent signal from the cell nuclei of the skin of the auricle of transgenic mice of the C57BL/6J-Tg(CMV-H2B-eGFP)12Ched line.

The invention is illustrated by the following examples.

Example 1. Obtaining a transgene cassette for microinjection

One microgram of H2B-GFP plasmid (Addgene #11680) was digested with AflII (NEB R0520S) and AseI (NEB R0526S) in 50 µl of 1x NEBuffer™ r2.1 buffer (NEB B6002S). The reaction was carried out at 37°C for 16 hours.

The reaction products were electrophoresed in a 1% agarose gel for 40 minutes with a DNA molecular weight marker, after which they were visualized using a UV transilluminator, and a DNA fragment of approximately 2000 bp was excised from the gel. The gel fragment containing the desired DNA fragment was then used for DNA extraction using the QIAquick Gel Extraction Kit (Qiagen 28704) according to the manufacturer's protocol. The DNA concentration in the resulting solution was determined using a Nanodrop 2000 and found to be 200 ng.

Example 2. Production of transgenic mice of the C57BL/6J-Tg(CMV-H2B-eGFP)12Ched line

C57BL mice were used in the study as oocyte and sperm donors, and CD-1 mice as recipients. Female oocyte donors, male sperm donors, and recipients were maintained in the SPF vivarium of the Institute of Cytology and Genetics SB RAS (ICG SB RAS) in individually ventilated cages (OptiRAT, Animal Care, USA) with wood shavings as bedding. The cages were maintained at an air temperature of 22-24°C and a 12:12 day:night cycle (morning at 8:00 a.m.), with 40-50% humidity and access to purified water enriched with mineral supplements (Severjanka, Ecoproject, Russia).

To obtain zygotes, oocyte donor females were superovulated using standard technology. 5-10 females were given 7.5 IU of pregnant mare serum gonadotropin (Folligon, MSD Animal Health, the Netherlands) intraperitoneally at 18:00 and 47 hours later 7.5 IU of human chorionic gonadotropin (hCG) (Chorulon, MSD Animal Health, the Netherlands) intraperitoneally. Then, 15 hours after the hCG injection, the oocytes in the form of cumulus oocyte complexes (COCs) were washed out of the Oviductal Ampula under mineral oil (FertiPro, Belgium) by incising it and then transferring the COCs into an HTF medium (pH = 7.2) warmed to 37 degrees Celsius, containing 21 mM HEPES buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); all further manipulations in atmospheric air were carried out in this medium. Then the COCs were transferred to a Thermo Scientific Midi 40 incubator (Thermofisher Scientific, USA), previously kept in CO ₂ , for at least 12 hours in the HTF medium for in vitro fertilization with 25 mM NaHCO ₃ (10). Then, about 100 thousand capacitated motile spermatozoa per oocyte were added to the COCs. Capacitation was carried out for one hour in the same medium used for fertilization. Four hours after fertilization, the cells were denuded, the presence of second polar bodies was assessed, and the fertilized oocytes were then transferred to a KSOM-AA medium (11) pre-incubated in _CO2 for at least 12 hours and coated with mineral oil. Further cultivation was carried out in this medium.

Culturing embryos after injection for 12 hours before transfer into a pseudo-pregnant female allows for a more accurate assessment of the effects of the injection and, as a result, the selection of the most viable embryos for transfer, compared to transfer immediately after injection.

The microinjection mixture was diluted in 50 mM TE buffer (pH 7.5) so that the concentration of the isolated fragment was 5 ng/μl. The microinjection system included an Olympus IX71 inverted microscope (Olympus, Japan) equipped with a condenser with DIC contrast and semi-apochromatic objectives LUCPLFLN40XRC, LUCPLFLN20XRC, CPLFLN10XRC (Olympus, Japan), as well as two MMO-4 three-axis oil-immersed hydraulic joysticks (Narishige, Japan): one for the injection needle and the other for the capillary for holding the cells.

For all studies, SPL plastic (SPL Life Sciences Co., Ltd, Korea), which passed the MEA test, was used.

Embryo transfer was performed as described previously (9), with minor modifications. Females were mated with verified vaectomized males; the day of detection of the vaginal plug (d.p.c.) was considered day 0 of pseudopregnancy. Females were anesthetized with 2.5% isoflurane at 0.5 d.p.c.; further manipulations with the animal were performed on a heated stage at 37°C. Two-cell embryos were transferred into the infundibulum of the oviduct.

Following the transfer of two-cell embryos, newborn mice were obtained. Mice carrying the CMV-H2B-eGFP transgene cassette were selected using nuclear fluorescence phenotyping. To obtain heterozygotes carrying the CMV-H2B-eGFP transgene cassette, the resulting mice were crossed with wild-type mice. The resulting heterozygous mice were then crossed to produce homozygous mice and the final line of mice transgenic for the CMV-H2B-eGFP cassette.

Example 3. Analysis of the presence of a transgene in the genome of the C57BL/6J-Tg(CMV-H2B-eGFP)12Ched transgenic mouse line

Genotyping for the presence of the CMV-H2B-eGFP construct was performed by detecting the GFP fluorescent signal from cell nuclei using a fluorescence microscope (Fig. 2).

For phenotyping of young animals, ear fragments obtained during standard animal tagging procedures upon separation from their mothers at three weeks of age were used. The ear fragments were analyzed using the ZOE imaging system in the eGFP fluorescence detection channel (Figure 2).

To identify homozygous carriers of the transgene construct, backcrosses were performed on wild-type C57BL/6 animals. In the case of homozygous carriers, 100% of the offspring exhibited phenotypic characteristics of CMV-H2B-eGFP cassette expression, while in the case of heterozygous carriers, only 50% did.

Thus, a new line of transgenic mice C57BL/6J-Tg(CMV-H2B-eGFP)12Ched was created, characterized by tissue-specific and age-dependent expression of the H2B-eGFP gene, namely its ubiquitous expression in perinatal age, and a decrease in expression by adulthood with the preservation of expression in highly specific cell types, mainly in endothelial cells (lining of blood vessels).

Sources of information

1. Wang S, Ren X, Wang J, Peng Q, Niu X, Song C, et al. Blocking autofluorescence in brain tissues affected by ischemic stroke, hemorrhagic stroke, or traumatic brain injury. Front Immunol. 2023 May 29;14:1168292.

2. Manzini G, Barcellona ML, Avitabile M, Quadrifoglio F. Interaction of diamidino-2-phenylindole (DAPI) with natural and synthetic nucleic acids. Nucleic Acids Res [Internet]. 1983 Dec 20 [cited 2024 Oct 25];11(24):8861-76. Available from: https://dx.doi.org/10.1093/nar/11.24.8861

3. Kanda T, Sullivan KF, Wahl GM. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Current Biology. 1998 Mar 26;8(7):377-85.

4. Musinova YR, Lisitsyna OM, Golyshev SA, Tuzhikov AI, Polyakov VY, Sheval E V. Nucleolar localization/retention signal is responsible for transient accumulation of histone H2B in the nucleolus through electrostatic interactions. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 2011 Jan 1;1813(1):27-38.

5. Hadjantonakis AK, Papaioannou VE. Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice. BMC Biotechnol [Internet]. 2004 Dec 24 [cited 2024 Oct 25];4:33. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC544401/

6. Kiyonari H, Kaneko M, Abe T, Shioi G, Aizawa S, Furuta Y, et al. Dynamic organelle localization and cytoskeletal reorganization during preimplantation mouse embryo development revealed by live imaging of genetically encoded fluorescent fusion proteins. Genesis [Internet]. 2019 Feb 1 [cited 2024 Oct 25];57(2):e23277. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC6590263/

7. Fraser ST, Hadjantonakis AK, Sahr KE, Willey S, Kelly OG, Jones EAV, et al. Using a Histone Yellow Fluorescent Protein Fusion for Tagging and Tracking Endothelial Cells in ES Cells and Mice. Genesis [Internet]. 2005 [cited 2024 Oct 25];42(3):162. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC1850986/

8. Luo C, Zuñiga J, Edison E, Palla S, Dong W, Parker-Thornburg J. Superovulation Strategies for 6 Commonly Used Mouse Strains. J Am Assoc Lab Anim Sci [Internet]. 2011 Jul [cited 2022 Sep 8];50(4):471. Available from: /pmc/articles/PMC3148645/

9. Nagy A, Gertsenstein M, Vintersten K, Behringer R. Manipulating the Mouse Embryo: A Laboratory Manual, Fourth edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 2014;814.

10. Kito S, Ohta Y. In vitro fertilization in inbred BALB/c mice I: Isotonic osmolarity and increased calcium-enhanced sperm penetration through the zona pellucida and male pronuclear formation. Zygote. 2008 Aug;16(3):249-57.

11. Ho Y, Wigglesworth K, Eppig JJ, Schultz RM. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol Reprod Dev [Internet]. 1995 [cited 2022 Sep 8];41(2):232-8. Available from: https://pubmed.ncbi.nlm.nih.gov/7654376/

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Claims (4)

1. A line of transgenic mice C57BL/6J-Tg(CMV-H2B-eGFP) expressing fluorescently labeled histone H2B at postnatal age, transformed with a transgene cassette consisting of a CMV promoter, a polyadenylation signal of the SV40 virus terminating transcription from this promoter, and an open reading frame of the chimeric H2B-eGFP gene with the sequence SEQ ID NO: 1. 2. A method for producing a line of transgenic mice according to claim 1, comprising microinjecting a solution of a transgene construct into the pronucleus of a fertilized egg of a donor mouse, identifying viable zygotes, transplanting two-cell embryos into pseudo-pregnant recipient females, obtaining newborn mice, selecting mice carrying the target mutation, crossing the latter with wild-type mice to obtain heterozygous females or mutant males carrying the target mutation, crossing heterozygous mice from the previous stage to obtain the final line of mice, characterized in that the transgene construct is a CMV-H2B-eGFP transgene cassette encoding a chimeric H2B-eGFP gene having the nucleotide sequence SEQ ID NO 1, wherein, before transferring the two-cell embryos into a pseudo-pregnant female, the latter are cultured for at least 12 hours in CO ₂ incubator on KSOM-AA nutrient medium under mineral oil. 3. The method according to claim 2, characterized in that the microinjection mixture is diluted in 50 mM TE buffer at pH 7.5 so that the concentration of the transgene cassette solution is 5 ng/μl. 4. The method according to paragraph 2, characterized in that the selection of mice carrying the target mutation is carried out using phenotyping based on the fluorescence of cell nuclei.