The high-level expression analysis of rhPA in the rhPA/gGH double-transgenic 2 rabbits and its thrombolysis activity in vitro

: 【 Objective 】 In this study, the rhPA/gGH double transgenic rabbits were constructed, and 19 the expression level of rhPA,rabbit growth and development features were analyzed, which might 20 provide a new idea for obtain rhPA high level expression transgenic animals. 【 Method 】 Two rhPA 21 transgenic rabbits fertilized eggs were microinjected with linearized GH plasmid to obtain the 22 rhPA/gGH rabbits.The integration of rhPA/gGH gene was detected by PCR. The rhPA expression 23 level in transgenic rabbit milk was detected by ELISA and Western blotting,and FAPA was 24 performed to detect the in vitro thrombolytic activity of rhPA.The body weight of transgenic rabbits at different growth stages were measured to test the effect of gGH gene on rhPA/gGH double 26 transgenic rabbits growth and development . 【 Result 】 A total of 151 rhPA transgenic rabbits fertilized 27 eggs were obtained through superovulation, 125 of them were microinjected with linearized GH 28 plasmid and transplanted into 8 surrogate mother rabbits.Six surrogate mother rabbits were pregnant, 29 with a pregnancy rate of 75.0% (6/8) ， 16 rhPA/gGH gene double transgenic rabbits were identified 30 by PCR (10♂,6♀). The rhPA expression levels in rhPA single-transgenic rabbit whey were 31 0.27–0.63g/L, while the rhPA expression leves were 4.98-12.24 g/L in the rhPA/gGH 32 double-transgenic rabbits whey. The rhPA expression levels of rhPA/gGH double-transgenic rabbit 33 whey were significantly increased by about 17.2–23.8 times, and had higher thrombolytic activity in 34 vitro. There was no significant difference in body weight between rhPA/gGH double transgenic 35 rabbits, rhPA single transgenic or non-transgenic rabbits from birthday to 10 months age(P>0.05). 36 【 Conclusion 】 The rhPA/gGH double transgenic rabbits were successfully constrected, which was 37 proved that the introduction of gGH gene could significantly increase the rhPA expression level in 38 the milk of transgenic rabbits and without affecting the growth and development of transgenic 39 rabbits, which laid a foundation for the preparation of transgenic rabbits with higher recombinant 40 protein expression level in the future, and also provide new ideas and new methods for the 41 establishment of mammary gland bioreactor.


Introduction 44
Thrombosis diease is a common and frequent disease that might seriously threaten human 45 health and life, and thrombolytic therapy is one of the most widely used and effective treatment 46 methods in clinical practice [1][2][3] . The human tissue-type plasminogen activator(tPA) is a serine 47 protease synthesized and secreted by vascular endothelial cells, which can efficiently and 48 specifically dissolve thrombus and it is a good second-generation thrombolytic drug [4][5] . The 49 recombinant human plasminogen activator (rhPA) in this study is a recombinant mutants of natural 50 tPA [6] , which belongs to the third generation thrombolytic drugs and has more superior thrombolytic 51 efficacy than natural tPA. Therefore, the study of how to stably improve the rhPA expression level is 52 an important guideline for the development of new thrombolytic drugs. 53 Currently, exogenous gene expression silencing is an important bottleneck in animal mammary 54 gland bioreactor research. Although some methods such as the use of friendly sites (Rosa26, Hipp11, 55 Pifs501), site-specific targeted integration (ZFNs, TALENs, CRISPR/Cas9), functional gene 56 recombination modification and optimization of cis-acting elements (promoters, introns, enhancers) 57 can overcome or alleviate gene expression silencing, there are still many limitations [11][12] . Therefore, 58 we need to study new ideal techniques to optimize and improve the exogenous genes expression 59 level of transgenic animal. At present, the strategies to improve the expression level of tPA and rhPA 60 gene are mainly in the modification of the gene itself and optimization of cis-acting elements. For 61 example, Ebert KM et al constructed transgenic goat to expression tPA in mammary gland with 62 active function at 3 μg/mL by modifying the tPA mutant recombinant [13] . Lu Y et al used sheep 63 β-lactoglobulin gene as a promoter regulatory sequence to construct the vector,by which the tPA 64 expressed in the mammary gland of transgenic mice was about 6 μg/mL [14] . Zhou Y et al used tPA to 65 replace the partial coding sequence of mouse whey acid protein to construct the mWAP-htPA hybrid 66 gene base, and the tPA expression level in the transgenic mice mammary gland was increased [15] . In 67 our laboratory, the goat β-casein and CMV were used as the hybrid promoter regulatory sequences 68 to construct a recombinant mammary gland-specific expression vector (PCL25/rhPA). The 69 expression level of recombinant tPA in the mammary gland of rhPA transgenic rabbits could reach 70 630μg/mL [16][17] . However, the expression level of tPA and rhPA in the above studies is still at a low 71 level and has never been scientifically and effectively solved. 72 Some researchers have proved that transgenic animals constructed through double gene 73 co-integration can produce synergistic effect, where one gene can promote the expression level of 74 the other gene and increase the expression level of the target gene [18][19][20] , resulting in higher yields. 75 For example, Sendtner M et al [19] found that transfection the double gene with ciliary neurotrophic 76 factor (CNTF) and leukemia inhibitory factor (LIF) was able to significantly increase the expression 77 level of LIF protein and produce trophic physiological effects on motor neurons. Chen  The gene fragments were diluted to 5 ng·μL -1 using TE buffer (5 mmol·L -1 Tris, pH 7.4 0.1 mmol·L -1 128 EDTA) and stored at -20°C. 129 130

Construction of rhPA/gGH transgenic rabbits 131
The rhPA transgenic rabbits (K29, K34) selected as donors and FSH were injected 132 intramuscularly into the hindlimbs muscle with 10 IU/each rabbit in the morning and evening (12 h 133 interval) for 3 d. On the 4th day, 5 IU/each FSH was injected intramuscularly at 7:00 a.m and 100 134 IU/each hCG was injected intravenously into the ear margins at 19:00 p.m.to obtain fertilized eggs. 135 On the 5th day at 12:00 noon, fertilized eggs were collected by surgery [16] .The rabbits were 136 anesthetized with subcutaneous atropine 1 mg·kg -1 and intravenous injection of zoletil-50 7.5 137 mg·kg -1 at the ear margin.The gGH gene fragments microinjected fertilized eggs were incubated in 138 an at 38°C, 5% CO2 saturated humidity incubator for 30 min. Then the fertilized eggs were 139 transplanted into the oviducts of synchronized estrous recipient female rabbits with 10-30 eggs each 140 to be pregnant. The process of transgenic rabbit surgery was shown in Figure 2. 141 142

PCR detection of transgenic rabbits 143
The ear tip tissue of newborn rabbits was cut aseptically about 1-2 mm 3 , and added tissue lysate 144 containing with 250 μg proteinase K, which digested overnight at 55°C. The genome was extracted 145 by phenol/chloroform extraction and precipitated by 100% ethanol for PCR detection.Two pairs of 146 primers for both rhPA and gGH genes were designed (as shown in Figure 1 and Table 1

), in which 147
CtPA-F/R primers were used for rhPA gene detection, and the PCR procedures were: 94℃ 148 pre-denaturation for 5 min; 94℃ denaturation for 1 min, 50℃ annealing for 45s, 72℃ extension for 149 45s, a total of 30 cycles; 72℃ extension for 5 min. CgGH-F/R primers were used for gGH gene 150 detection, and the PCR parameters were: 94℃ pre-denaturation for 5min; 94℃ denaturation for 151 1min, 54℃ annealing for 45s, 72℃ extension for 1min, a total of 30 cycles; 72℃ extension for 5min. 152 PCR amplification products were subjected to 1% agarose gel electrophoresis to determine whether 153 the band size was correct. 154 Transgenic female rabbits were mated with male rabbits and collected milk. The milk was 158 centrifuged at 10,000×g for 30 min to remove the upper fat layer and the lower turbid layer, then the 159 whey was diluted 100 times in PBS for detection.100 μL whey and 100 μL coating buffer (1.696 160 g·L -1 Na2CO3, 2.856 g·L -1 NaHCO3, pH 9.6) were added to each well in 96-well ELISA plate 161 overnight at 4°C. The coating buffer was discarded and washed 3 times with PBS containing 0.05% 162 Tween-20.Add 200 μL of sealing fluid (PBS containing 10% fetal bovine serum)to each well and 163 incubated at 37 ℃ for 2 h. The mouse anti-tPA monoclonal antibody was used as the primary 164 antibody (sc-59721, Santa Cruz), and the goat anti-mouse monoclonal antibody IgG-HRP was used 165 as the secondary antibody (sc-2005, Santa Cruz).All of the antibody was incubated respectively at 166 37 ℃ for 2 hours. 50 μL of chromogenic reagent (5 mg OPD, 15 μL 30% H2O2,28.4 g·L -1 Na2HPO4, 167 19.2 g·L -1 citric acid) was added to each well and incubated in dark at 37°C for 20min. After 168 coloration, the OD450 value was measured by microplate reader. Alteplase was used as a standard to 169 draw a standard curve,then calculate rhPA expression level in rhPA/gGH double-transgenic rabbits 170 and rhPA single-transgenic rabbits. 171 172

FAPA assay for thrombolytic activity 187
Using PBS buffer as solvent, 1% agarose gel, 10mg/mL fibrinogen and 10U/mL thrombin were 188 prepared respectively. Boil and melt 1% agarose gel, and take 20mL in a 50mL centrifuge tube, until 189 the temperature dropped to about 50°C without scalding. Warm 1mL of 10mg/mL fibrinogen 190 preheated to 37℃ and add to agarose gel,then take 1mL of 10U/mL thrombin preheated to 42℃ and 191 add to agarose gel. After agarose gel solidification at room temperature, punch and seal the bottom, 192 and added 50 μL of rabbit whey into each well,alteplase as a positive control, normal non-transgenic 193 rabbit whey as a negative control, and PBS as a blank control. The wells were placed at 37℃ 194 overnight to measure the sizes of the transparent rings.  Table 2. 231  The body weights of transgenic rabbits integrated with rhPA single gene and rhPA/gGH double 272 gene were measured continuously from month age up to the 10th month and compared with those of 273 normal non-transgenic rabbits (Table 3). There were no significant difference in body weights at 274 different growth stages, which were not statistically significant (P > 0.05). In addition, the monthly 275 body weight of all rabbits increased significantly from 0 to 6 months, and there were significant 276 difference in monthly body weight (P<0.05). After 7 months, the rabbits monthly weight difference 277 were not significant (P>0.05). The growth curves of rabbits (Figure 7) weight showed that the 278 introduction of gGH gene did not affect the normal growth and development of rabbits. Compared 279 with rhPA single-transgenic rabbits and normal non-transgenic rabbits, the weight growth trend of 280 rhPA/gGH double-transgenic rabbits was consistent. After 7 months, the weight growth tends to be 281 gentle, and the average weight of rabbits grown to 10 months were between 4.5-5.0 kg. It can be 282 found when rabbits grew to adulthood (10 months old), there were no significant difference in body 283 weight between rhPA/gGH double genes, rhPA single transgenic rabbits and normal non-transgenic 284 rabbits (P>0.05) (Fig.8). The results indicate that the transfer of gGH gene did not affect the normal 285 growth and development of the transgenic rabbits, and the rhPA/gGH double transgenic rabbits were 286 able to survive and grow normally to adulthood. 287 Table 3 Weight measurement of normal rabbits and transgenic rabbits at different growth 288 stages(x± s) 289 According to the statistical analysis of the World Health Organization (WHO), the annual 292 number of deaths due to cardiovascular diseases is about 13 million worldwide, among which 293 thrombotic diseases account for more than 50%, and there is an obvious increasing trend [28][29] . At 294 present, thrombolytic drugs such as alteplase (tPA), reteplase, monteplase, lanoteplase, and 295 tenecteplase are mainly used in clinical treatment of thrombosis [30] . The recombinant human 296 fibrinogen activator (rhPA) in this study is a newly developed third-generation recombinant 297 thrombolytic drug with the advantages of high efficiency, safety, specificity, small side effects and 298 so on. The clinical use of thrombolytic drugs is mostly produced by prokaryote or mammalian cell 299 expression, which has the limitations of low production or high price, and the popularization of mass 300 use has been limited [16,31]. Therefore, how to efficiently and conveniently produce rhPA at low cost 301 and higher activity has always been a hot topic in scientific research. Since the successful expression 302 of human α-antitrypsin in sheep mammary gland by Wright et al in the 1990s [32] , mammary gland 303 bioreactors have shown an attractive prospect, which provides a great possibility for the production 304 of recombinant thrombolytic drugs. However, rhPA and tPA is a non milk protein, and its expression 305 levels in animal mammary glands is low [13][14][15][16][17] . Therefore, it is particularly important to explore how 306 to improve the expression level of non-lactoprotein rhPA in animal mammary glands. 307 At present, there are many methods to improve the efficiency of exogenous gene expression in 308 animals [11][12]33] . The two genes in double transgenic organisms can produce synergistic promotion to 309 regulate the gene network system of the organism, and to increase the expression level of exogenous 310 target genes [34] . In recent years, there have been many reports on double genes to improve the 311 expression level of exogenous target genes. For example, Kundu S et al [21] introduced 312 NUP98-PHF23 (NP23) and NUP98-HOXD13 (NHD13) gene expression vectors into mice to 313 prepare NP23-NHD13 double transgenic mice, and successfully expressed high-level target genes. 314 The studies of Chen XY et al [20] and Sendtner M et al [19] on pig and human somatic cells also 315 proved the phenomenon of double gene pro-expression, which led to a significant increase in the 316 expression level of target genes. Therefore, double gene synergistic pro-expression is a good strategy 317 to increase target gene expression, which provides a new idea for improving the expression of rhPA 318 level in transgenic rabbit mammary glands. 319 Since 1920, when EVANS first demonstrated that the growth-promoting substance in the 320 pituitary gland is growth hormone, scholars have studied the GH gene extensively and intensively, 321 and have achieved important results [35] . It has been reported that growth hormone (GH) is able to 322 combine with the HRE sequence of the β-casein gene to promote receptor activation and 323 synergistically increase the specific expression of lactoproteins [25,36] . Therefore, it is highly possible 324 to improve the expression level of exogenous genes in transgenic animal mammary gland by using 325 GH gene introduction. However, the current strategy to improve the expression level of tPA and 326 rhPA genes in the mammary gland of transgenic animals is often to optimize the gene vector 327 construction [15][16][26][27] . There are few reports on the synergistic promotion of tPA gene expression by 328 double transgenic animals, especially the study of GH gene synergistically promoting the expression 329 of tPA in transgenic animals at home and abroad. 330 Rabbit is one of the most widely used experimental animals in life science. It is also a model 331 organism commonly used in transgenic experiments. Compared with large animals such as cattle or 332 goat, it has the advantages of more ovulation, short pregnancy, strong fecundity and more estrus 333 throughout the year. Compared with mice, it has the advantages of high lactation and suitable for the 334 production of recombinant medical proteins, which can fill the "blank" between large and small 335 animals [16,37] . The rhPA single transgenic rabbits (goat β-casein gene as the regulatory sequence and 336 verified expression) were selected as the donor rabbits [16] , and 151 fertilized eggs were obtained by 337 superovulation via FSH/hCG. The gGH gene was injected into the pronucleus of fertilized egg by 338 microinjection, and then transplanted into the synchronous estrus female New Zealand recipient 339 rabbits, respectively. Forty rabbits were successfully delivered. Sixteen rhPA/gGH double transgenic 340 rabbits (10♂,6♀) were obtained by PCR integration detection, and the double gene integration rate 341 was significantly better [13][14][15][16][17][26][27] . 350 In addition, many studies on GH transgenic animals have focused on the ability of growth 351 hormone to regulate the growth of the organism, resulting in a "super" species with an individual 352 size exceeding that of the general wild type [42] . However, the rhPA/gGH double transgenic rabbits 353 obtained in this study showed that the gGH gene did not affect the growth and development of 354 transgenic rabbits by comparing their growth and development with normal non-transgenic rabbits, 355 and the transgenic rabbits with integrated gGH were able to grow and develop normally into 356 adulthood. In general, the body weight of New Zealand adult rabbits is 4.0-5.0 kg [43] . In our study, 357 the six double transgenic rabbits were continuously monitored for 10 months, and it was found that 358 there was no significant difference in body weight between the transgenic rabbits,rhPA transgenic 359 and normal non-transgenic rabbits at different stages of growth and development. The body weights 360 were 4.5-5.0 kg at the age of 7-10 months, and the weight growth was not obvious. It was 361 speculated that the transgenic rabbits had the same growth pattern as the normal rabbits. The analysis 362 of the results proved that the gGH introduced in this experiment did not affect the growth and 363 development of the transgenic rabbits. The reason may be due to the fact that the gGH selected for 364 the experiment derived from goats rather than rabbits, which could not produce physiological effects 365 similar to those in goats and did not affect the growth and development of the rabbits. Moreover, the 366 gene expression is a multifaceted effect involving integration sites, epigenetics, copy number of 367 exogenous gene, relevant hormone levels and gene networks [44][45][46] . Therefore, the related studies still 368 need to be continued. 369