GFP

GFP

GENERATION OF A STABLE TRANSGENIC SWINE MODEL FOR CELL TRACKING AND CHROMOSOME DYNAMICS STUDIES

Renan B. Sper1,2, Sean Simpson1,2, Xia Zhang1,2, Bruce Collins1,2, Jeff Sommer 3 Bob Petters3,Jorge A. Piedrahita1,2

1Department of Molecular Biomedical Sciences, 2Center for Comparative Medicine and Translational Research, 3 Department of Animal Science, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA.

Results

Figure 1. (A) Squematic representation of knockin strategy into the 3’UTR of the beta actin locus via

CRISPR-CAS9 system. Drawing shows the actin genomic locus and the donor plasmid containing the

IRES-H2B-GFP flanked by two homology arms of 1Kb each. (B) Porcine fibroblast colonies heterozygous

(male) for the knockin were utilized as donor cells for somatic cell nuclear transfer. Generated transgenic

embryos were transferred to a recipient sow. Three viable piglet (males, P1 generation) were obtained and

use to generate F1 generation via artificial insemination of wild type sows (3 pregnancies obtained).

Introduction Over the last few years transgenic pigs have become an attractive research model in the

field of translational research, regenerative medicine, and stem cell therapy due to their

anatomic, genetic and physiological similarities with humans. The development of

transgenic murine model via fusion of GFP to Histone 2B protein (H2B, a protein of

nucleosome core) allows easier and convenient methods of tracking cell migration and

engraftment levels after transplantation and a opportunity to better understand the

complexity of chromosome dynamics during cell cycle. Up to date, the development of a

stable transgenic swine model expressing H2B-GFP has not been described. Such model

would have multiple applications in the field of stem cell/regenerative medicine. The

objectives of this study include: 1) Developing a viable H2B-GFP transgenic swine model

via CRISPR-CAS9 recombination into the ACTB locus (β actin) and Somatic Nuclear

Transfer (SCNT), and :2) Demonstrating that H2B-GFP fetal liver hematopoietic stem

cells are capable of engrafting porcine severe combined immunedeficient (SCID) fetuses

(42 days) via ultrasound guided in utero hematopoietic stem cell transplantation (IUHCT).

The hypothesis for the described study include: 1) CRISPR-CAS9 driven targeted

integration of IRES-H2B-GFP would allow the development of the first transgenic swine

model with ubiquitous expression of chromatin-bound GFP and :2) H2B-GFP fetal liver

hematopoietic stem cell can be tracked and analyzed when transplanted via IUHCT into

42 days SCID fetuses, reversing the immunedeficient phenotype at birth, resulting in

fully differentiated GFP positive leukocyte in peripheral blood and lymphoid tissue.

B

GENERATION OF β ACTIN H2B-GFP TRANSGENIC PIGS

Material and Methods

Conclusion CRISPR-CAS9 is capable of driving H2B-GFP transgene into β actin locus via homologous recombination, resulting in the

generation of the first described H2B-GFP swine model with ubiquitous expression of chromatin-associated GFP.

Intranuclear GFP allows chromosomes dynamic to be visualized in vivo and in vitro.

H2B-GFP gene transmission on F1 generation was 55.8% (in concordance with Mendel's Law). P1 and F1 generation

animals are healthy and viable at this point.

H2B-GFP fetal liver hematopoietic stem cells are capable of engrafting SCID transgenic developing fetuses via IUHCT and

reversing immunedeficient phenotype. Engrafted cells can be easily tracked via flow cytometry analysis of peripheral blood

and via fluorescence microscopy of lymphoid tissue.

A

A

Figure 4. (A) Flow cytometry analysis of peripheral blood of 80 days old wildtype and SCID H2B-GFP engrafted pigs. When

staining for pig CD45 and pig CD3 (signal on x axis), a distinct GFP + population is evident (y axis).(B) Left image shows 2x

H&E slide of spleen from SCID H2B-GFP engrafted pigs showing tissue architecture. Right image shows Optimal Cutting

Temperature (OCT) frozen section from spleen of same pig. Two X image shows GFP fluorescence microscopy demonstration

multiple white pulp regions containing H2B-GFP engrafted cells (periarteriolar lymphoid sheaths). Forty X image shows blue

circled region amplified and stain for DAPI, engrafted cells demonstrate DAPI and GFP signal while host cells stain for DAPI

only.

H2B-GFP P1 generation H2B-GFP F1 generation

3’ UTR

E1 E2 E3 E4 E5 E6 PolyA signal

* *

IRES-pH2B-eGFP LHa RHa

IN UTERO H2B-GFP HEMATOPOIETIC STEM CELL TRANSPLANTATION INTO 42 DAYS SCID FETUSES

Figure 2 (A) Day 42 H2B-GFP fetal liver hematopoietic stem cell were isolated and cryopreserved until

transplantation. (B) Five randomly picked swine H2B-GFP postive fetuses were selected as donors for

IUHCT, and 5 42 days transgenic SCID fetuses were injected with 3.5 million viable cells/each via

ultrasound guided fetal intrahepatic injection for engraftment. Four viable fetuses were delivered

naturally.

A

B

UBIQUITOUS EXPRESSION OF H2B-GFP TRANSGENIC PIGS

C

Figure 3. A shows intranuclear H2B-GFP expression in OCT-embedded intestines, muscle and skin for DAPI, GFP

and merge (40X) . B shows fluorescence microscopy (40x) of in vitro cultured bone marrow derived mesenchymal

stem cells from H2B-GFP F1 generation, chromosome dynamics through GFP allows cell cycle staging (a cell in

Telophase stage is shown). C shows bright field and GFP images of 8 days in vitro cultured SCNT H2B-GFP

blastocyst (20x ).

H2B-GFP CELL TRACKING INTO SCID PIGS POSTNATALLY

ANTI CD3

A

GF

P

B

WT STAINED PIGLET A STAINED PIGLET B STAINED

Spleen H&E 2x

Spleen GFP 2x

IUHCT day 42 SCID H2B-GFP engrafted pig

H2B-GFP fetal liver hematopoietic stem cells

GFP

Results

40x GFP+DAPI merge

B intestines

DAPI GFP Merge

muscle

DAPI GFP Merge

skin

DAPI GFP Merge ANTI CD45