The effect of leptin transgenic Plasmodium yoelii on mouse body weight

Abstract

Abstract:

Objective To investigate the effect of leptin transgenic Plasmodium yoelii on mouse body weight. Methods To construct the leptin gene-containing CRISPR/Cas9 recombinant plasmid which had the 5′UTR and 3′UTR of MIF（macrophage migration inhibitory factor） of Plasmodium yoelli 17XNL strain at two ends, the exogenous mouse leptin gene was inserted downstream of MIF coding region through homologous recombination, resulting in the PYC-MIF-Leptin recombinant plasmid. The recombinant plasmid was then electroporated into P.y 17XNL mature schizonts, and the transgenic schizonts were used to infect a Kunming mouse via tail vein injection. The trangenic P.y clone was screened by pyrimethamine selection and identified by PCR. The trangenic or wild-type P.y was used to infect a C57BL/6 mouse respectively. Blood sample was collected through eye ball and tail vein, and immunofluorescence and RT-PCR were performed to determine the expression of leptin protein in the parasites. Finally, PBS （200 μl） containing trangenic or wild-type P.y （1 × 10⁴） was injected through the tail vein into C57BL/6 mice（n = 5 respectively）. The negative control received a same volume of PBS. The changes of parasitemia and body weight were recorded every two days. Results The leptin-expressing recombinant plasmid PYC-MIF-Leptin was constructed successfully. Results of DNA sequencing of transgenic parasites confirmed the integration of leptin gene at the downstream of MIF gene and successful transcription. Immunofluorescence results indicated successful expression of mouse leptin protein. The weight loss was significant in mice infected with transgenic parasites on day 17（17.26 ± 1.40）g, decreased by 10.7%, but not in the other two groups. Both transgenic and wild-type parasites began to decline when parasitemia reached about 10%, but the transgenic parasites proliferated more rapidly. Both disappeared at 23 days. Conclusion Infection with leptin transgenic parasites decreases the body weight of the infected mice.

Key words: CRISPR/Cas9 technique, Transgenic, Plasmodium yoelli 17XNL, Leptin, Weight loss

CLC Number:

R382.319

Kai LI, Hong ZHENG, Feng ZHU, Yong FU, Wen-yue XU. The effect of leptin transgenic Plasmodium yoelii on mouse body weight[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2017, 35(1): 36-41.

Figures/Tables 8

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig.4

Fig. 5

Fig. 6

Fig. 7

References 15

[1]	Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue[J]. Nature, 1994, 372(6505): 425-432.
[2]	Kokot F, Adamczak M, Wiecek A, et al.Does leptin play a role in the pathogenesis of essential hypertension[J]. Kidney Blood Press Res, 1999, 22(3): 154-160.
[3]	Meister B, Arvidsson U.The hormone leptin reduces body weight. A mutant gene makes the mouse obese[J]. Lakartidningen, 1996, 93(4): 247-251.
[4]	Drbalova K.Leptin--the key to obesity[J]. Cas Lek Cesk, 1998, 137(12): 355-358.
[5]	Bolze F, Morath V, Bast A, et al. Long-acting pasylated leptin ameliorates obesity by promoting satiety and preventing hypometabolism in leptin-deficient lep(ob/ob) mice[J]. Endocrino-logy, 2016, 157(1): 233-244.
[6]	Kissileff HR, Thornton JC, Torres MI, et al. Leptin reverses declines in satiation in weight-reduced obese humans[J]. Am J Clin Nutr, 2012, 95(2): 309-317.
[7]	Landau I, Chabaud AG.Natural infection by 2 plasmodia of the rodent thamnomys rutilans in the central African Republic[J]. C R Acad Sci Hebd Seances Acad Sci D, 1965, 261(1): 230-232.
[8]	付雍, 丁艳, 周桃莉, 等. 约氏疟原虫17XL和17XNL感染小鼠组织病理学差异研究[J]. 成都医学院学报, 2011, (2): 111-114.
[9]	Jinek M, Chylinski K, Fonfara I, et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 2012, 337(6096): 816-821.
[10]	Zhang C, Xiao B, Jiang Y, et al. Efficient editing of malaria parasite genome using the CRISPR/Cas9 system[J]. MBio, 2014, 5(4): e1414.
[11]	Ghorbal M, Gorman M, Macpherson CR, et al. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system[J]. Nat Biotechnol, 2014, 32(8): 819-821.
[12]	宋盟. 鼠疟原虫TatD_likeDNase序列的表达与分析[D]. 长春:吉林大学, 2013.
[13]	齐洁, 张军, 任慧娴, 等. 血传、蚊传保种对约氏疟原虫毒力的影响及对其机制的探讨[J]. 第三军医大学学报, 1991, 13(3): 265-269.
[14]	李孟英, 彭恒, 陈要朋, 等. 田鼠巴贝虫病的诊治及文献复习[J]. 中华实验和临床感染病杂志(电子版), 2014, 8(3): 332-335.
[15]	Fox BA, Bzik DJ.De novo pyrimidine biosynthesis is required for virulence of Toxoplasma gondii[J]. Nature, 2002, 415(6874): 926-929.