bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1

Highly efficient transgenic mouse production using2

piggyBac and its application to rapid phenotyping at

3

the founder generation

4

Eiichi Okamura1*, Seiya Mizuno2*#, Shoma Matsumoto1*, Kazuya Murata2, Yoko Tanimoto2,

5Dinh Thi Huong Tra2, Hayate Suzuki2, Woojin Kang2, Tomoka Ema2, Kento Morimoto3, 4,

6

Kanako Kato2, Tomoko Matsumoto5, Nanami Masuyama6, 7, 8, Yusuke Kijima6, Toshifumi

7

Morimura1, Fumihiro Sugiyama2, Satoru Takahashi2, Eiji Mizutani9, Knut Woltjen5, Nozomu

8

Yachie6, 8, 10, 11, Masatsugu Ema1#

91

Department of Stem Cells and Human Disease Models, Research Center for Animal Life Science,

10

Shiga University of Medical Science, Otsu, Shiga, Japan

11

2

12

Tsukuba, Ibaraki, Japan

13

3

14

University of Tsukuba, Tsukuba, Ibaraki, Japan

15

4

Research Fellow of the Japan Society for the Promotion of Science, Chiyoda-ku, Tokyo, Japan

16

5

Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto

17

University, Sakyo-ku, Kyoto, Japan

18

6

19

University of British Columbia, Vancouver, British Columbia, Canada.

20

7Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata, Japan.

218

Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan.

22

9Laboratory of Stem Cell Therapy, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki,

23

Japan

24

10

25

Tokyo, Japan.

Laboratory Animal Resource Center, Trans-border Medical Research Center, University of Tsukuba,

Doctoral Program in Medical Sciences, Graduate School of Comprehensive Human Sciences,

School of Biomedical Engineering, Faculty of Applied Science and Faculty of Medicine, The

Research Center for Advanced Science and Technology, The University of Tokyo, Meguro-ku,

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26

11

27

Suita, Osaka, Japan28* These authors contributed equally to this work.

Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Osaka University,

29
30#Correspondence should be addressed to:31

Seiya Mizuno, Laboratory Animal Resource Center, Trans-border Medical Research Center,

32

University of Tsukuba, Tsukuba, Ibaraki 305-8575, Japan

33

Tel.: +81-29-853-3393

34

Email: konezumi@md.tsukuba.ac.jp

3536

Masatsugu Ema, Shiga University of Medical Science, Seta, Tsukinowa-cho, Otsu, Shiga 520-2192,

37

Japan

38

Tel.: +81-77-548-2334

39

Email: mema@bella.shiga-med.ac.jp

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40

Abstract

41

Pronuclear microinjection is the most popular method for producing transgenic (Tg) animals.

42

Because the production efficiency is typically less than 20%, phenotypic characterization of Tg

43animals is generally performed on the next generation (F1) onwards. However, apart from in rodents,

44

in many animal species with long generation times, it is desirable to perform phenotyping in the45

founder (F0) generation. In this study, we attempted to optimize a method of Tg mouse production to46

achieve higher Tg production efficiency using piggyBac transposon systems and established optimal

47

conditions under which almost all individuals in the F0 generation were Tg. We also succeeded in48

generating bacterial artificial chromosome Tg mice with efficiency of approximately 70%. By

49

combining this method with genome editing technology, we established a new strategy to perform50

phenotyping of mice with tissue-specific knockout using the F0 generation. Taking the obtained

51

findings together, by using this method, experimental research using Tg animals can be carried out

52

more efficiently.

53

Keywords: transgenic, piggyBac, transposon, conditional knockout

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54

Introduction

55

Transgenic (Tg) technology enables genetic engineering techniques to introduce foreign DNA

56

sequences, namely transgenes from other species or artificial ones, into the genomes of organisms.

57

This technology can be used to produce organisms that express useful traits, such as resistance to58

infectious diseases, a high growth rate, and herbicide resistance. In the medical field, Tg technology

59

has also made significant contributions, including the creation of disease model animals and the60

elucidation of gene function1–4. Another method for inserting foreign DNA into the genome is the61

knock-in (KI) approach, which involves inserting foreign genes into specific targeted locations, in

62

contrast to Tg technology in which genes are inserted into the genome at random. Although KI

63technology has the advantage of being able to perform genetic modification in a predictable manner,64

it is generally inefficient and has difficulty introducing large-sized DNA. These problems with KI

65

technology are being overcome in some animals such as mice and rats via the development of66

genome editing technology5–7. However, Tg technology is still considered useful for introducing

67

foreign genes into large animal species, including rabbit, pig, cattle, and non-human primates,

68

because efficient KI technology for such purposes has not yet been established.

69

The first Tg animal reported globally was a Tg mouse created by Jaenisch and Minz in the70

1970s8. A retrovirus was used as a vector for producing this mouse, but the transgene was not

71

expressed persistently due to silencing. In 1980, a pronuclear microinjection method, which is72

currently the most popular technique for producing Tg mice, was reported9. However, the efficiency

73

of generating Tg mice using this method is approximately 20% at best10. Therefore, obtaining

74

enough Tg mice in the F0 generation is difficult, and thus phenotypic analysis is generally performed

75

using animals from the next generation onwards. The efficiency of Tg animal production should thus76

be improved in order to enhance research efficiency and to reduce the number of animals used, from77

the perspective of animal welfare. A more efficient method for generating Tg mice is a method using78

lentivirus first reported in 200311, in which Tg mice can be produced with approximately 80%–100%

79

efficiency. However, the lentiviral method is associated with the limitation that it can introduce

80transgenes of approximately 8 kbp at most.

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81

Another method for generating Tg animals is the transposon-based method. Transposons are82

DNA sequences that can move from one location to another within the genome of an organism, and83

were originally found in maize12. During the transposition, the transposase enzymes bind to the84

inverted terminal repeats (ITRs) that define the boundaries of the transposon and cut out the DNA

85

sequence between ITRs from the genome and insert it into another location. Several

86

transposon-based methods have been established and used to produce Tg animals, including Tol213,

87

Sleeping Beauty14, and the piggyBac15 system. Transposon-based methods can introduce large

88transgenes of 100 kbp or more, but the Tg production efficiency is generally less than 45%16, which89

is lower than for the lentiviral method.

90

Recent developments in genome editing technology have made it possible to very efficiently

91

produce systemic knockout (KO) animals, in which large-scale phenotypic screening analysis at the92

F0 generation can be conducted17. Approximately 23% of gene KOs result in embryonic lethality18, in

93

which case tissue-specific KOs are often required to explore gene functions in a given tissue.

94

However, the establishment of tissue-specific Cre Tg mice and flox mouse strains is time-consuming95

and laborious. Although many such mouse strains have been created and useful bioresources have96

been established, tissue-specific KO often takes more than a year to obtain, cross, and finally obtain97

the next generation for phenotypic analysis. Therefore, several methods have been reported to98

perform tissue-specific KO in a single generation19–25, but each method has its own advantages and99

disadvantages, and thus they should be used appropriately depending on the purpose.

100

Here, we report a novel strategy for generating tissue-specific KO called ScKiP (single-step101

cKO mouse production with piggyBac). We increased the efficiency of generating Tg mice with the102

piggyBac transposon system by optimizing the method to deliver transposase and donor DNA into103

the mouse zygote, and succeeded in producing Tg mice with an efficiency of nearly 100%. This104

method even enabled us to introduce an approximately 170 kbp bacterial artificial chromosome

105(BAC) vector with an efficiency of approximately 70%. By using this more efficient method, we

106

succeeded in establishing a new approach to performing tissue-specific KO at the F0 generation. This107

approach is expected to contribute to animal welfare by reducing the number of animals used andbioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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also improve the efficiency of research.

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109

Materials and methods

110

Study approval

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All animal experiments were carried out in accordance with the Fundamental Guidelines for Proper

112

Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the113

jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology, and Guidelines114

for Proper Conduct of Animal Experiments from the Science Council of Japan. All animal

115

experimental procedures were approved by the Animal Care and Use Committee of Shiga University

116

of Medical Science (2020-6-21) and the Institutional Animal Experiment Committee of the117

University of Tsukuba (22-019).

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119

Mice

120

C57BL/6J and ICR mice were purchased from Charles River Laboratories (Yokohama, Japan).

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PWK/Phj mice (RBRC00213) were obtained from RIKEN BioResource Research center (Tsukuba,

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Japan). The mice were maintained in plastic cages under pathogen-free conditions in a room

123

maintained at 23.5 ± 2.5°C and 52.5 ± 12.5% relative humidity under a 14 h light:10 h dark cycle.

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125

Establishment of CdEC flox mouse line

126

We selected a sequence (5¢-CGC CCA TCT TCT AGA AAG AC-3¢) located in the first intron of the127

Gt(ROSA)26Sor as the CRISPR target. Briefly, an expression cassette consisting of CAG promoter,128

floxed EGFP, and Cas9NmC was placed between the 5¢-homology arm and the 3¢-homology arm. A

129

rabbit beta-globin polyadenylation signal sequence was placed downstream of each of floxed EGFP

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and Cas9NmC. Cas9NmC is a fusion protein of Cas9 and part of mouse Cdt1 that we previously

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reported26. The CRISPR–Cas9 ribonucleoprotein complex and donor DNA were microinjected into

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zygotes of C57BL/6J mice, in accordance with our previous report27. Subsequently, microinjected

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zygotes were transferred into the oviducts of pseudopregnant ICR female and newborns were134

obtained. Genotyping for confirmation of the KI allele and random integration allele was performed

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using the same methods as in our previous report26. The CdEC flox mouse line was deposited to136

RIKEN BioResource Research center (RBRC12114).

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138

In vitro fertilization, electroporation, and microinjection

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Fertilized eggs were obtained by in vitro fertilization using a method described elsewhere28. Briefly,

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oocytes were collected from oviducts of 10–14-week-old C57BL/6J females superovulated by the141

intraperitoneal administration of CARD HyperOva (Kyudo, Tosu, Japan), followed by human142

chorionic gonadotropin (hCG). Sperm were collected from the caudal epididymis of 12–16-week-old

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C57BL/6J males, and then preincubated in Fertiup Mouse Sperm Preincubation Medium (Kyudo).

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Insemination was performed in CARD medium (Kyudo), followed by incubation at 37 °C in an

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atmosphere containing 5% CO2 for 3 to 6 h. Fertilized eggs were washed to remove cumulus cells

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and sperm, and incubated in KSOM medium (Ark Resource, Kumamoto, Japan) until

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electroporation. Electroporation of fertilized eggs was performed in Opti-MEM I medium containing

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piggyBac mRNA using Super Electroporator NEPA21 (Nepa Gene, Ichikawa, Japan) as described149

elsewhere29 with minor modifications. In this study, the poring pulse was set as follows: 225V, 2 ms

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pulse width, 50ms pulse interval, 4 pulses, 10% attenuation rate, and + polarity. Meanwhile, the151transfer pulse was set as follows: 20V, 50ms pulse width, 50ms pulse interval, 5 pulses, 40%

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attenuation rate, and ± polarity. The eggs were incubated until pronuclei were clearly visible. The

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transposon DNA was microinjected under a microscope equipped with a micromanipulator and154

Femtojet (Eppendorf, Hamburg, Germany). The injected one-cell embryos were cultured in KSOM

155

medium until the two-cell stage and then transferred into pseudopregnant ICR mice.

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157

In vitro transcription

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The template plasmid for piggyBac transposase (mPBase) for in vitro transcription was made by159

replacement of a mouse codon-optimized mPBase cDNA sequence with an EGFP sequence of

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pcDNA3.1-EGFP-poly(A)30. After linearization of this template plasmid using XhoI restriction

161

enzyme, mPBase mRNA was synthesized using the mMESSAGE mMACHINE T7 ULTRA

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Transcription Kit (AM1345; Thermo Fisher Scientific, Waltham, MA, USA) or mMESSAGE

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mMACHINE T7 Transcription Kit (AM1344; Thermo Fisher Scientific).

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165

BAC DNA recombination

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A BAC DNA clone (RP24-125B24) containing the mouse Flk1 locus was obtained from Invitrogen

167(Invitrogen, Carlsbad, CA, USA). Modification of the BAC DNA to insert the GFP sequence into the168

first exon of the Flk1 gene was performed in a previous study31,32. To insert ITR (inverted terminal

169

repeat) sequences and Kusabira-Orange expression cassette into the backbone vector region, the170

BAC DNA was further modified by the same method as described in that previous study31 using the171

RED/ET recombination technique (Gene Bridges, Heidelberg, Germany). Collected recombinants172

were identified by screening for kanamycin resistance, followed by PCR analysis. The PGK-gb2-neo

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expression cassette was not excised.

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175

Genotyping of the embryo

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E11.5 (embryonic day 11.5) stage embryos were collected from the uteri of surrogate mothers after177

euthanasia and the placenta and yolk sac were removed. The fluorescent signal was captured under a178

fluorescent stereoscopic microscope (FL5; Leica Microsystems, Wetzlar, Germany) equipped with a179

camera (DP73; Olympus, Shinjyuku, Japan). Genomic DNA from the embryos was extracted by180

phenol/chloroform extraction. Genotyping PCR was performed using a primer set to detect the GFP

181

sequence using conventional PCR (T100; Bio-Rad, Hercules, CA, USA). To determine the copy

182

number of the transgene, droplet digital PCR (ddPCR) was performed using the Qx200 Droplet

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Digital PCR system (Bio-Rad) with the GFP and Tfrc Primer/Probe set.

184
185

Southern blotting

186

Genome DNA was extracted from mouse embryos with phenol and chloroform, and then purified by187

ethanol precipitation. After the digestion of 5µg of genomic DNA by EcoRI, the concentration was

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measured using a Quantus Fluorometer (Promega, Madison, WI, USA). One microgram of digested

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DNA was electrophoresed in 1% agarose gel. The DNA was blotted onto a nylon membrane

190(Amersham Hybond-N+; GE Healthcare, Chicago, IL, USA) and hybridized with DIG-labeled probe

191

using DIG Easy Hyb (Roche Diagnostics, Mannheim, Germany). Salmon sperm DNA was used in192

the blocking step. After the reaction with anti-digoxigenin-AP Fab fragments (Roche), the bands

193

were detected by enhanced chemiluminescence using CDP-Star (Roche) and ImageQuant LAS 4000

194

Mini (GE Healthcare). All probes were synthesized by PCR using DIG DNA Labeling Mix, 10´

195Conc. (Roche).

196
197

Inverse PCR

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PCR was performed using a method described elsewhere33 with minor modifications. In this study,

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3µg of DNA was digested by EcoRI and the digested DNA was self-ligated using DNA Ligation Kit

200(Mighty Mix; Takara Bio, Kusatsu, Japan) after purification by ethanol precipitation. Regions where201

transgenes became integrated were amplified using transgene-specific primers by shuttle PCR.

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Nested PCR was also performed to enrich the genomic regions of interest.

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204

Echocardiography

205

A Vevo 2100 High-Resolution Imaging System (Visual Sonics Inc, Toronto, ON, Canada) was used206

to evaluate cardiac contractility. Mice were anesthetized with 2.5% isoflurane in oxygen and placed207

on a warmed platform. The isoflurane concentration was maintained at 1.5% during imaging. An

208ultrasound gel was applied to the left anterior thorax after the removal of fur from the body.

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Short-axis M-mode images were obtained at the level of the papillary muscle using a 40-MHz

210transducer. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic

211

diameter (LVESD) were measured using Vevo LAB software (FUJIFILM Visualsonics, Toronto, ON,

212

Canada). Fractional shortening (FS) was calculated at three different time points of each mouse as213

follows: FS (%) = (LVEDD – LVESD)/LVEDD × 100.

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Results

215

piggyBac transgenesis by electroporation and pronuclear microinjection

216

Although previous works achieved considerably high efficiency in the generation of Tg mice15, we

217

assumed that Tg mice could be generated with even higher efficiency by initiating the expression of218

mammalian codon-optimized mPBase at an earlier timing than in the previous methods. To test this,219

we introduced in vitro-synthesized mPBase mRNA into zygotes by electroporation early after in220

vitro fertilization (Figure 1a). Consistent with previous reports describing that excessive amounts of

221

mPBase inhibit transposition activity, a phenomenon called overproduction inhibition (OPI), we

222

confirmed that OPI occurred at a high dose of mPBase in mouse ES cells, by varying the amount of223

mPBase expression plasmid with a certain amount of donor plasmid DNA (Figure 1b). To optimize

224

the concentration of mPBase mRNA for electroporation, we applied it at 0, 20, 100, and 500ng/µl.

225

After electroporation, the fertilized eggs were cultured in vitro, and donor plasmid DNA containing a

226

GFP expression cassette (Figure 1c) was microinjected into the pronucleus at the PN3–4 stage

227(Figure 1a). Those embryos were cultured in vitro until the E4 stage. We counted the blastocysts

228

exhibiting GFP fluorescence under each set of conditions by fluorescence microscopy and found that229

conditions with 500ng/µl mRNA gave the best results, at which approximately 80% of the230

blastocysts exhibited fluorescence (Figure 1d, e). To confirm this result at a later developmental

231

stage, we transplanted two-cell-stage embryos into the oviduct of a surrogate mother, removed

232

embryos from the uterus at E11.5 and dissected them, and conducted fluorescence observation as

233

well as ddPCR to detect the transgene. The obtained results indicated that, for the 100ng/µl group, 9234

out of 10 were Tg, while for the 500 and 1000ng/µl groups, all embryos were Tg. Although there

235

were no significant differences in copy number of the transgene among all groups, the 500ng/µl

236

group had the highest mean copy number (Figure 1f). From these results, we concluded that an

237mRNA concentration of 500ng/µl is optimal to produce Tg mice.

238

To confirm that this method would result in healthy births, we transplanted the fertilized eggs

239

into surrogate mothers. We found that, out of 70 fertilized eggs, 12 (17%) animals were born; ofbioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

240

these, 1 animal was eaten by its mother, while the rest of them (11 animals) were Tg (Figure 1g). To

241

evaluate the reproductive ability of the F0 generation, three males were mated and all resulting

242

offspring were found to be Tg (Figure 1h).

243
244

Bacterial artificial chromosome DNA integration by piggyBac transposase

245

Relatively short plasmids were used as the donor DNA in our assay, and therefore we attempted to246

use larger donor DNAs with several expression cassettes. We constructed two plasmids with a larger

247

12kbp transgene. One was a construct containing an expression cassette for GFP-P2A-mCherry248

linked to the Ubiquitin C promoter (Figure 1i). All eight E11.5 embryos showed GFP and mCherry

249

expression (Figure 1j). The other was a construct containing expression cassettes for GFP linked to250

the Myh6 promoter and tdTomato linked to the CAG promoter (Figure 1k). We obtained four251

embryos that reached the E9.5 stage, seven that reached E11.5, and three newborns, all of which252

showed tdTomato expression (Figure 1l&m). We also confirmed heart-specific GFP expression in all

253of the embryos at E9.5 and E11.5 by fluorescence microscopy (Figure 1l&n). Finally, ddPCR was

254

performed using DNAs extracted from E11.5 embryos to analyze the transgene copy number. The

255

results revealed 9.7±8.6, and 5.8±3.3 copies of the transgenes in UbC pro.-GFP-P2A-mCherry and

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Myh6 pro.-GFP-CAG-tdTomato mice, respectively (Figure 1o).

257

Mice of 12kbp Tg were established at 100% efficiency, and therefore we applied this method to258

establish BAC Tg mice because the efficiency of BAC Tg mouse production is typically very low. In

259

a previous study, we constructed BAC DNA with a GFP sequence linked to the Flk1 promoter. Here,260

the ITR sequences and Kusabira-Orange cassette were inserted into the vector backbone of the BAC

261

DNA (Figure 1p). The engineered BAC DNA was used as a donor DNA at two different

262

concentrations (1 and 10ng/µl). Owing to the high viscosity of BAC DNA, microinjection at263

concentrations higher than 10ng/µl was not feasible. We obtained 14 Tg animals out of 19 embryos

264

at E11.5 under conditions with 1ng/µl, while 7 out of 10 embryos were Tg at E11.5 under conditions

265

with 10ng/µl only when mPBase had been introduced (Figure 1q). Thus, there was no significant

266

difference in the percentage of Tg mice under the two sets of conditions. Next, we evaluated thebioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

267

transgene copy number in these Tg mice by ddPCR and found that the mean copy numbers were268

0.39 and 2.39 for 1 and 10ng/µl, respectively, which were significantly different (Figure 1r). The

269

BAC Tg embryos exhibited ubiquitous Kusabira-Orange expression throughout the body and270

specific GFP signals in the blood vessels (Figure 1s), consistent with a previous report31. Finally,271

PCR genotyping analysis indicated that the percentage of Tg mice among the offspring was very

272high (52 out of 94; 55%).

273
274

Donor DNA injection into fertilized eggs refrigerated after electroporation

275

Although the method using electroporation and microinjection developed in this study can produce276

Tg mice with very high efficiency, the experimental schedule is inconvenient due to microinjection

277

being performed late at night. Therefore, we modified the schedule by storing the eggs after

278

electroporation in a fridge, and then performed microinjection the next morning (Figure 2a). After

279

either a mixture of BFP/EGFP/tdTomato expression vectors (three colors) or a mixture of

280

BFP/EGFP/tdTomato/E2Crimson (four colors) (Figure 2b) was microinjected, the fertilized eggs281

were transplanted into the oviduct of surrogate mothers at the one-cell stage. We dissected the282embryos at E14.5 and checked the presence of fluorescence as an indicator of Tg. We obtained 12283

embryos, 11 (91.7%) of which were Tg under the three-color conditions, while 3 out of 4 embryos

284(75%) were Tg under the four-color conditions (i.e., at least one fluorescence signal was shown)

285(Figure 2c&d). These results indicate that Tg mice can be generated at high efficiency even after286

refrigeration of the fertilized eggs.

287
288

Rapid phenotyping of conditional KO mice at the F0 generation by ScKiP

289

Currently, for phenotypic analysis of essential genes in a certain tissue that play roles in embryonic290

survival, the Cre-loxP system must be used, which is time-consuming and laborious. By using the291

method established in this study, we attempted to establish a new strategy to generate conditional292

KO at the F0 generation by using piggyBac transgenes. First, we targeted the Prmt1 gene, which293

causes dilated cardiomyopathy of young mice upon heart-specific KO, while its systemic KO isbioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

294

embryonically lethal34. For heart-specific KO, we constructed a donor plasmid with the Cre gene295

linked under a cardiac-specific Myh6 promoter and a gRNA for the Prmt1 gene expression cassette

296(Figure 3a). Prior to the conditional KO experiment, to check that the Myh6 promoter causes Cre

297

expression in the heart, we produced Tg mice in the GRR mouse35 strain background. The GRR

298

allele has a structure in which the floxed EGFP-polyA sequence and the tdTomato gene are linked

299

under the CAG promoter, and tdTomato is expressed following Cre-mediated excision (Figure 3b).

300

The donor plasmid was microinjected into the pronuclei after storage at 4°C overnight, and those301

eggs were transplanted into the oviduct of a surrogate mother at the one-cell stage. We obtained 14

302

offspring, 10 of which were revealed to possess the transgene (GRR KI/Tg+) by genotyping PCR

303

analysis. Fluorescent microscopy of the born mice indicated that most of the GRR KI/Tg+ mice304

showed strong tdsRed fluorescence in the heart, along with nonspecific expression in regions

305

including the skin (Figure 3c&d). To perform conditional KO, we used the CdEC flox mouse strain

306

containing the KI allele in which the floxed EGFP-polyA sequence and the Cas9NmC gene are307

linked under the CAG promoter, and Cas9NmC is expressed only after Cre expression (Figure 3e).

308

Tg mice with the same donor vector were produced in a similar way in the CdEC flox mouse strain.

309

The results revealed that 10 offspring were obtained, and genotyping PCR analysis revealed that all

310

animals possessed the CdEC flox KI allele, while only one had the transgene (CdEC KI/Tg+).

311

Echocardiography at 2, 4, and 6 weeks old revealed that cardiac contractility of the CdEC KI /Tg+

312

mouse was reduced compared with that in mice without transgene (CdEC KI/Tg-, Figure 3f). At 50

313

days old, we noticed that the CdEC KI/Tg+ mouse was sluggish and wheezed, and thus euthanized it.

314

The heart of this CdEC KI/Tg+ mouse weighed 188mg, which was markedly heavier than that of a315

CdEC KI/Tg- mouse (101mg, Figure 3g), suggesting dilated cardiomyopathy similar to that of316

conventional cKO mice reported previously34. Thus, although we were able to observe a phenotype

317

of heart-specific KO of the Prmt1 gene at the F0 generation, the rate of Tg mice relative to all318

offspring was low at 10% in this experiment. This may have been due to ectopic Cre expression319

other than in the heart in Tg embryos, resulting in embryonic lethality.

320

Given the success of characterizing the heart-specific phenotype associated with Prmt1, webioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

321

applied this strategy to characterize mice with germ cell-specific KO of the Kit gene at the F0322

generation. It has been reported that mice with hypo-functional Kit point mutant (WV/WV) had no323

sperm, while systemic KO with deletion of the transmembrane domain (W/W) resulted in perinatal-324

or late fetal-stage lethality. To avoid lethality due to non-specific Cre-loxP recombination, we used

325

germ cell-specific Ddx4 promoter-driven CreERT2, which works only in the presence of tamoxifen

326

as a component of the donor plasmid with an expression cassette of gRNA for the Kit gene (Figure3273h). Similar to the procedure in the previous experiment, after mPBase introduction into fertilized

328

eggs prepared using CdEC flox KI male and wild-type female mice, the donor plasmid was

329

microinjected into the pronuclei, followed by transplantation into the oviduct of a surrogate mother330

at the one-cell stage. We obtained 14 offspring, including 11 males and 3 females (Figure 3i), all of

331

which were revealed to have the transgene and KI allele (CdEC KI/Tg+) by genotyping PCR. Nine

332

males were used for subsequent experiments. From each of these nine males, one testis was removed333

as a control (before tamoxifen sample) at 16 to 17 weeks old. Tamoxifen was injected three times at334

18, 22, and 23 weeks old to induce Cre-loxP recombination. During this process, six mice died335

probably due to tamoxifen toxicity, postoperative complications, or non-specific Kit gene KO in336

cells other than germ cells. At 32 weeks old, the remaining three males were euthanized and their337

testes were collected (after tamoxifen sample). HE-stained sections from the removed testes were338

prepared and the proportion of seminiferous tubules not containing sperm heads was determined

339(Figure 3j). It was found that the number of seminiferous tubules without sperm increased after340

tamoxifen administration compared with that before tamoxifen administration in all three males

341(Figure 3k). When the percentage of Cas9-expressing undifferentiated spermatogonia after342

tamoxifen administration was examined by immunostaining using anti-Cas9 and anti-GFRa1

343

antibody, reductions in undifferentiated spermatogonia (8.5%, 86%, and 59%) were seen in samples

344

No. 1, 2, and 3, respectively (Figure 3l). We named this cKO method “ScKiP,” which stands for345

single-step cKO mouse production with piggyBac.

346
347

Preference of the Tg integration site and parental alleles in mouse zygote

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

348

To reveal the preference of integration sites into which Tg is introduced by our piggyBac system, we349

prepared fertilized eggs with PWK/Phj (PWK) strain sperm and C57BL/6J (B6) strain oocyte, and350

then injected mPBase mRNA and a donor plasmid DNA containing GFP and a DNA barcode351

sequence (Figure 4a). We obtained 17 E11.5 embryos, 9 of which were Tg as judged by fluorescence352

microscopy and genomic PCR. We investigated the number of integration sites in each Tg embryo

353

by Southern blotting (Figure 4b). We confirmed that one to three transgenes had integrated into

354

different genomic regions of the genome in each embryo. We further performed ddPCR analysis to355

evaluate the copy number (Figure 4c). Subsequently, we detected the DNA barcode sequence of the356

Tg by Amplicon-seq; overall, we detected 16 unique DNA barcode sequences. To investigate the357

sites of integration of the Tg in the genome, we performed inverse PCR and confirmed the results by358

genotyping PCR using primers specific for the DNA barcode sequence. Focusing on the genomic

359

features of the integration site, 9 out of 16 transgene were located in introns, 3 were in repeat

360

sequences, 3 were intergenic regions and only 1 was located coding sequence. (Figure 4d). Using

361

information on the SNPs between the PWK and B6 genomes, we could determine whether the362transgene was introduced into the paternal or maternal allele for 13 out of 16 transgenes (Figure 4e).

363

Overall, 3 transgenes were on the maternal allele, even though we injected donor DNA into the male364

pronucleus. This suggested that the DNA integration event by mPBase continued after pronuclear

365

fusion. To obtain a deeper understanding of the integration sites, we performed comparative analysis

366

using ChIP-seq data. It was reported that the integration sites for mPBase were enriched in regions

367

with H3K4me336. We downloaded H3K4me3 ChIP-seq data of the zygote and early two-cell embryo

368with PWK sperm and B6 oocyte from the GEO database, and compared the peaks with the369

integration sites (GSE71434)37. Overall, 31.25% of integration sites overlapped with H3K4me3,

370

which is generally located on the active TSS, for the integrated allele (Figure 4f, g).

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

371

Discussion

372

In this study, we established a highly efficient Tg production method using the piggyBac transposon373

system. Three types of transposon systems are commonly used to create Tg animals: piggyBac,

374

Sleeping Beauty, and Tol238. Among them, we employed piggyBac because it is the only transposase375

used to create Tg mice with transgenes larger than 150 kbp39. By mPBase mRNA electroporation and376

pronuclear donor DNA microinjection, a plasmid-sized transgene with 100% efficiency and a BAC

377

transgene with approximately 70% efficiency could be introduced.

378

Previous works indicated that the efficiency of Tg mouse production by mPBase was typically379

20%–70%. The previously described methods and ours differ in terms of how mPBase is380

introduced. In several previous studies, mPBase expression plasmid vector was introduced into the381

cytoplasm of unfertilized eggs or the pronucleus of fertilized eggs15,40–42. Urschits et al. showed that

382

mPBase protein expression was at the background level until 6 h after injection of the expression

383

plasmid, and peak expression was observed at about 30 h40. Therefore, mPBase is fully expressed384

after the two-cell stage, and donor plasmids introduced at the one-cell stage might be degraded or

385

diluted until the mPBase protein functions. In other reported studies, mPBase mRNA was used386

instead of an expression plasmid, but it was injected at the pronuclear stage43–47, and thus full

387

expression may have occurred after the two-cell stage. In contrast, in this study, we attempted to388

introduce mPBase mRNA immediately after fertilization using electroporation. In this method,

389

mPBase was sufficiently expressed when donor DNA was injected into the pronucleus and the390

transgene integration may have occurred efficiently before donor DNA degradation or dilution.

391

In this study, although we attempted to improve the efficiency of piggyBac transgenesis in mice,392

improvement of Tg production efficiency is also useful for other animal species. In rat, another393

popular experimental animal, pronuclear injection of a piggyBac expression plasmid vector and394

donor DNA has been used to generate Tg animals, but the efficiency was generally less than395

30%44,48,49. Although a study reported that Tg rats can be produced more efficiently (33%–100%,396

mean of nearly 80%) using mPBase mRNA pronuclear microinjection50, it may be possible to397

consistently produce Tg animals even more efficiently if mPBase mRNA is introduced at an earlierbioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

398

timing after in vitro fertilization. Transgenic technology is also important for livestock, such as cattle,

399

pig, and sheep, but there are very limited reports on application of the piggyBac transposon system

400

to their zygotes51,52. Somatic cell nuclear transfer is one of the most common methods to create Tg

401livestock53,54. However, this approach is technically difficult, and the birth rate is typically low.

402

Although no reports have yet been published on the use of mPBase mRNA injection to produce Tg

403

livestock, this approach is worth pursuing.

404

Mice with tissue-specific conditional KO are very useful to elucidate gene functions in vivo but

405

are time-consuming to produce because they typically require flox and Cre-Tg animal strains to be406

obtained and crossed. Recently, new methods have been developed to perform tissue-specific KO in407

a rapid manner. One example involves introducing CRISPR-Cas9 ribonucleoproteins into mice using408

nanoparticles, which enables efficient KO in several tissues, including liver, muscle, brain, kidney,409

and lung19–22. Another example involves introducing the flox allele into zygotes obtained from410

tissue-specific Cre-Tg mice by KI technology23. A third example involves the introduction of gRNA411

by an adeno-associated virus (AAV) vector into tissue-specific Cas9-expression Tg mice. Finally, a412

method has been developed involving the introduction of gRNA and a tissue-specific Cre expression

413

vector by AAV into mice harboring a Cre-loxP reaction-dependent Cas9 expression cassette.

414

Although these methods are very useful, they vary in terms of their targetable tissues, efficiency of415

cKO generation, and procedural difficulty. Thus, it is important to carefully examine these options

416

and choose an appropriate method considering the purpose of the planned research. In this study, as

417

another option to perform cKO experiments within a short time, we developed the ScKiP method418

and tested its usefulness. For rigorous evaluation, we selected Prmt1 and Kit as target genes, for419

which a cKO experiment is necessary to reveal their functions after birth because their systemic KO

420

is embryonically lethal. Although we confirmed that this method is not strictly tissue-specific,

421

probably due to positional effects, we could observe the expected phenotype. Therefore, the ScKiP

422

method is considered to be useful for rapid screening analysis of gene function. In addition, using the423

ScKiP method, KO cells can be identified using the disappearance of EGFP fluorescence as an424

indicator, which is a feature not found in other methods described above.

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

425

Taking the obtained findings together, by using the Tg production method that we have426

established, we can obtain a sufficient number of animals for phenotypic analysis in the F0427

generation. Our methods are expected to contribute to animal welfare by reducing the number of428

animals used and enabling research to be carried out more efficiently.

429
430

Acknowledgments

431

pcDNA3.1-EGFP-poly(A) plasmid was kindly provided by Dr. Kazuo Yamagata (KINDAI

432

University). This work was supported by JSPS KAKENHI Grant Numbers 21K05988, 23H03860 (to433E.O.), 20K22611 (to S.M.), and 21H02388 (to M.E.). We thank Central Research Laboratory at

434

Shiga University of Medical Science and Single-cell Genome Information Analysis Core (SignAC)

435

at WPI-ASHBi, Kyoto University, for their support. We also thank Edanz (https://jp.edanz.com/ac)

436

for editing a draft of this manuscript. Finally, we thank all members of the Department of Stem Cells

437

and Human Disease Models, Research Center for Animal Life Science, Shiga University of Medical

438

Science.

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

439

Figure legends

440

Figure 1 Generation of Tg mice by mPBase mRNA electroporation and donor plasmid DNA

441

pronuclear microinjection

442(a) Schematic representation of the method to introduce mPBase mRNA and donor plasmid DNA.

443

Typical schedule for performing each procedure is shown at the top of the figure. (b) Donor plasmid

444

introduction into the ES cell genome using the piggyBac transposon system. ES cells were445

transfected with different concentrations of the mPBase expression plasmid vector and the same446

concentration of the donor plasmid, and the number of puromycin-resistant colonies was counted. (c)447

Schematic representation of CAG-EGFP-IRES-Puro donor plasmid DNA. Only the region between

448

two ITRs is described. (d, e) Investigation of the optimal concentration of mPBase mRNA by449

observing blastocyst embryos using fluorescence microscopy. Fluorescence microscopic images of450

conditions with mRNA at 0 and 500ng/µl are shown in d. Tg embryos were judged by the presence451

of GFP fluorescence (orange arrow: GFP positive, white arrow: GFP negative blastocysts), with

452

GFP-positive rates being displayed as a bar graph in e. Blue dots indicate blastocyst development453

rates for the number of fertilized eggs tested. (f) Investigation of the optimal concentration of454

mPBase mRNA using DNAs from E11.5 embryos. Transgene copy numbers were determined by

455

ddPCR for the GFP sequence. (g) Fluorescent image of born Tg mice at the P1 (postnatal day 1)

456stage. (h) A mating test was performed to confirm germline transmission of the transgene. Three Tg

457

male mice (#8, #9, #10) were crossed with wild-type (WT) females; the pedigree of line #8 is shown.

458

Regarding line #8, copy numbers of the transgene were determined by ddPCR using DNA obtained459

from the tail tip. The numbers in black boxes indicate the copy numbers of the transgene determined

460

by ddPCR using tail-derived DNA from F0 and F1 animals. (i) Schematic representation of UbC

461

pro.-GFP-P2A-mCherry donor plasmid DNA. Only the region between two ITRs is described.

462

cHS4: chicken hypersensitive site-4 insulator element. (j) Fluorescent images of E11.5 embryos

463

harboring UbC pro.-GFP-P2A-mCherry transgene. Bright field (left), GFP fluorescence (middle),464

and mCherry fluorescence images (right) are shown. (k) Schematic representation of Myh6465

pro.-GFP-CAG-tdTomato donor plasmid DNA. Only the region between two ITRs is described. (l)bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

466

Fluorescent images of representative E9.5 embryos harboring Myh6 pro.-GFP-CAG-tdTomato

467

transgene. Bright field image taken using a stereomicroscope (left), tdTomato fluorescence (2nd from

468

left), GFP fluorescence (2nd from right), and merged images (right) taken using a confocal

469

microscope are shown. (m) Fluorescent images of E11.5 embryos harboring Myh6

470

pro.-GFP-CAG-tdTomato transgene. Bright field (left) and tdTomato fluorescence images (right) are

471

shown. (n) Confocal microscopic images of representative E11.5 embryos harboring Myh6

472

pro.-GFP-CAG-tdTomato transgene. Transverse frozen sections were prepared. Bright field image473

taken using a phase contrast microscope (upper left), tdTomato fluorescence (upper right), GFP

474

fluorescence (lower left), and merged images (lower right) taken using a confocal microscope are475

shown. (o) Copy number analysis of Tg embryos harboring UbC pro.-GFP-P2A-mCherry transgene

476(UbC) and Myh6 pro.-GFP-CAG-tdTomato transgene (Myh6). (p) Schematic representation of BAC

477

DNA used as a donor vector. (q) Investigation of the optimal concentration of donor DNA using

478

DNAs from E11.5 embryos. Transgenes were detected by conventional PCR and GFP-positive rates479

are displayed as a bar graph. Blue dots indicate E11.5 development rates for the number of480

transplanted two-cell-stage embryos. (r) Copy number analysis of BAC Tg. Transgene copy numbers

481

were determined by ddPCR for the GFP sequence using DNAs from E11.5 embryos. (s) Fluorescent482

images of a representative E11.5 BAC Tg embryo. Bright field (left), Kusabira-Orange fluorescence483(middle), and GFP fluorescence images (right) of the whole body (top), head (middle), and somite

484(bottom) region are shown. The red rectangular frames inside the whole body represent the head and485

somite region.

486
487

Figure 2 Generation of Tg mice using fertilized eggs refrigerated after electroporation

488(a) Schematic representation of method to introduce mPBase mRNA and donor plasmid DNA.

489

Fertilized eggs were temporarily refrigerated after electroporation. Typical schedule for performing

490

each procedure is shown at the top of the figure. (b) Schematic representation of BFP, EGFP,

491

tdTomato, and E2Crimson expression cassettes on the donor plasmids. (c) Summary of the results of492

observing the obtained embryos by fluorescence microscopy. The presence of each fluorescencebioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

493

color is indicated by + and its absence is indicated by -. The results of three- (top) and four-color

494

donor plasmid injection (bottom) are shown separately. (d) Fluorescent images of representative495

E11.5 embryo harboring four-color transgenes. BFP (left), EGFP (2nd from left), tdTomato (2nd from496

right), and E2Crimson fluorescent images (right) are shown.

497
498

Figure 3 Rapid phenotyping of conditional KO mice499(a) Schematic representation of Myh6-Cre-Prmt1 gRNA donor plasmid DNA. Only the region

500

between two ITRs is described. cHS4: chicken hypersensitive site-4 insulator element. pA:

501

polyadenylation signal. (b) Schematic representation of GRR KI allele. (c) Fluorescent images of502born GRR KI mice with Myh6-Cre-Prmt1 gRNA transgene (GRR KI/Tg+) at the P4 (postnatal day

5034) stage. Fluorescent image of GRR KI mice without the transgene (GRR KI/Tg-) was shown as a504

control. (d) Fluorescent images of heart from GRR KI/Tg+ and GRR KI/Tg- mice. (e) Schematic

505

representation of CdEC flox KI allele. (f) Results of echocardiography on 2-, 4-, and 6-week-old

506

CdEC flox KI mouse with Myh6-Cre-Prmt1 gRNA transgene (CdEC KI/Tg+) or without transgene

507(CdEC KI/Tg-). (g) Morphogenic observation of hearts from 6-week-old CdEC KI/Tg+ and CdEC

508

KI/Tg- mice. (h) Schematic representation of gRNA-Ddx4-CreERT2 donor plasmid DNA. Only the509

region between two ITRs is described. (i) Observation of newborn CdEC KI mice with the510gRNA-Ddx4-CreERT2 transgene (CdEC KI/Tg+). (j) Results of HE-staining analysis of testes

511

removed from CdEC KI/Tg+ mice before and after tamoxifen injection. Arrowheads indicate

512

seminiferous tubules not containing sperm heads. (k) Percentage of seminiferous tubules without

513

sperm head in the testes from CdEC KI/Tg+ mice before and after tamoxifen injection. (l) Results of514

the immunostaining analysis of testes from CdEC KI/Tg+ mice after tamoxifen injection using515

anti-Cas9 and anti-GFRa1 antibody.

516
517

Figure 4 Generation of Tg mice using oocytes from C57BL6J and sperm from PWK strain

518(a) Schematic showing the timing of donor DNA microinjections relative to cell-cycle progression in519

the mouse embryo. (b) Results of Southern blot analysis using DNA from E11.5 Tg embryo obtainedbioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

520

with a probe for the GFP region. DNA from WT mouse was used as a negative control. DNA from521

GRR KI mouse was used as a positive control. (c) Copy number analysis of the transgenes using

522

DNA from E11.5 Tg embryo obtained by ddPCR with a probe for the GFP region. (d) Summary of523

genome-wide distribution of the transgene insertion sites. (e) Summary of the transgene integration

524

alles. (f) Percentage of transgene insertion sites overlapping with H3K4me3 peaks. (g) An example

525

of a transgene insertion region overlapping with H3K4me3 peaks.

bioRxiv preprint doi: https://doi.org/10.1101/2023.12.10.570953; this version posted December 10, 2023. The copyright holder for thispreprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

526

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Figure 1
PM 3:00

PM 9:00

AM 10:00

IVF

mPBase mRNAelectroporation

Donor DNA
microinjection

Transplantation

Bright Fieldc

5.1 kbpe

GFP

mPBase mRNA concentration
500 ng/ul
0 ng/ulViability (%)

mPBase experssion plasmid (ng)gfCopy number of transgenedNumber of ES colonies / wellb

AM 9:00

Percentage of
GFP positive embryos (%)

a

Tg

h

#8
F0 13.2

WT#8-1 #8-2 #8-3 #8-4 #8-5 #8-6 #8-7

mPBase mRNA (ng/μl)i

WTmPBase mRNA (ng/μl)

F1 8.7

4.9

13.9

7.1

7.9

3.9

Flk1 locus (167 kbp)p
11.8 kbp
GFP

mCherry

Bright field

GFP

Merge

n

Bright field tdTomato

tdTomato
GFP

Merge

o

Donor DNA (ng/μl)
mPBase mRNA

r

-

+

SomitetdTomato

Copy number of transgene

Bright field

Copy number of transgenem11.7 kbp

Viability (%)

klsPercentage of
GFP-positive embryos (%)

q

Whole body

Bright field

Head

j

Donor DNA (ng/μl)

Bright field

Kusabira Orange

GFP

6.7

Figure 2
AM 9:00a

PM 3:00

PM 8:00

AM 9:00

4℃

mPBase mRNAelectroporation

IVF

b

CAG

BFP

3’ITR

5’ITR

CAG

GFP

3’ITR

5’ITR

CAG

tdTomato

3’ITR

5’ITR

CAG

E2Crimson

3’ITR

4.2 - 4.9 kbp

PM 1:00

Donor DNA
microinjection

Transplantation

37℃

Store at 4℃

Recovery from 4℃c
4 colors
3 colors

5’ITR

AM 10:00

d

BFP

EGFP

tdTomato

E2Crimson

Figure 3b GRR Knock-in

e CdEC flox Knock-in
Rosa26Rosa26

CdEC KI/Tg+ (n=1)

GRR KI/Tg+

GRR KI/Tg-

CdEC KI/Tg -

No. 1

No. 2

i

No.3

l

k
Seminiferous tubules
without sperm heads (%)

BeforeTamoxifen
After
Tamoxifen

CdEC KI/Tg- (n=3)

GRR KI/Tg-

8.5 kbp
No.2

f

loxP

GRR KI/Tg+

Cas9

h

No expression

GFRa1

d

No.1

loxP

Not heart specific expression

expression

j

loxP

DAPI GFRa1 Cas9

c Heart specificloxP

9.2 kbp

Fractional shortening (%)

a

g

2w

4w

6w

CdEC KI/Tg +No. 3

Figure 4
a

b
#1

#2

PWK x B6 F0 Tg#4 #5 #6 #7#3*

Paternal(PWK)*

Maternal(B6)*
G1 phase

S phase

c

d

#9

*

*

*

*

*

*

*
*

WT GRR***

Donor DNA
Injection#8**

G2 phasee

Total (n=16)f

Total (n=16)

Total (n=16)

Copy number of transgene

6.25 %
18.75 %
(3)

(1)

18.75 %
(3)

18.75 %
(3)

Genomic feature

56.25 %
(9)

CodingIntronic
RepeatIntergenic

18.75 %
(3)

Integrated allele
62.5 %(10)

Gm5444 locus (mm9)g

1 kb
4,788,500

chr13:
H3K4me3

Gm5444
paternal (PWK)
maternal (B6)
paternal (PWK)
early 2-cell maternal (B6)
Zygote4,789,000

Integration site
intron (1/2)

4,789,500

paternal allele
maternal allele
not determined

31.25 %
(5)68.75 %
(11)

Overlap with H3K4me3
not overlap